RESUMO
Trimetazidine, an antiischaemic drug, has been shown to restore impaired mitochondrial functions. Specific binding sites for [(3)H]-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 (K(i)=7 nM) for one of these classes of sites. Compounds from different pharmacological classes were evaluated for their ability to inhibit [(3)H]-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 [(3)H]-trimetazidine sites with sigma receptors. [(3)H]-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 [(3)H]-trimetazidine binding. Ca(2+) was the most potent inhibitor and totally suppressed the binding of [(3)H]-trimetazidine to the sites of medium affinity. An endogenous cytosolic ligand was able to displace [(3)H]-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 [(3)H]-trimetazidine binding sites could have a physiological relevance and be involved in the antiischaemic effects of the drug.
Assuntos
Mitocôndrias/metabolismo , Receptores de Superfície Celular/efeitos dos fármacos , Trimetazidina/análogos & derivados , Trimetazidina/metabolismo , Vasodilatadores/metabolismo , Algoritmos , Animais , Sítios de Ligação/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Cátions/farmacologia , Citosol/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Ratos , Ratos WistarRESUMO
1. 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. 2. [3H]-trimetazidine binds to two populations of mitochondrial binding sites with Kd values of 0.96 and 84 microM. 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. 3. Drug displacement studies with pharmacological markers for different mitochondrial targets showed that [3H]-trimetazidine binding sites are different from previously described mitochondrial sites. 4. 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 microM. 5. 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. 6. A strong correlation between swelling inhibition potency and low-affinity [3H]-trimetazidine binding sites was observed: r=0.907 (n=24; P<0.001). 7. These data suggest that mitochondrial sites labelled with [3H]-trimetazidine may be involved in the MTP inhibiton.
Assuntos
Mitocôndrias Hepáticas/metabolismo , Trimetazidina/metabolismo , Vasodilatadores/metabolismo , Animais , Sítios de Ligação , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Masculino , Permeabilidade , Ratos , Ratos Wistar , Frações Subcelulares/metabolismo , TrítioRESUMO
1. 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. 2. 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 microM). 3. The effect of ZDV-3P on mitochondrial swelling required a preincubation time. When incubated 10 min with mitochondria, ZDV-3P (8 microM) totally inhibited the rate of swelling. 4. 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 microM, in the presence of 20 microM, ADP, ATP or atractyloside, respectively. 5. 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. 6. 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).
Assuntos
Antivirais/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Didesoxinucleotídeos , Relação Dose-Resposta a Droga , Masculino , Mitocôndrias Hepáticas/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
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.
Assuntos
Hepatócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Piperazinas/farmacologia , Substâncias Protetoras/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Hepatócitos/metabolismo , Isquemia/patologia , Isquemia/prevenção & controle , Hepatopatias/patologia , Hepatopatias/prevenção & controle , Testes de Função Hepática , Masculino , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/fisiologia , Dilatação Mitocondrial/efeitos dos fármacos , NADP/metabolismo , Piperazinas/uso terapêutico , Substâncias Protetoras/uso terapêutico , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controleRESUMO
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.
Assuntos
Cinarizina/farmacologia , Flunarizina/farmacologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Mitocôndrias Hepáticas/metabolismo , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Técnicas In Vitro , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , NADP/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Permeabilidade , Ratos , Ratos WistarRESUMO
The protective effect of N-[(3, 5-di-tertiobutyl-4-hydroxy-1-thiophenyl)]-3-propyl-N'-(2,3, 4-trimethoxybenzyl)piperazine (S-15176) on liver injury induced by warm ischemia-reperfusion was investigated using a rat model. Animals were subjected to 2 h of ischemia followed by different reperfusion times. Hepatocyte integrity was assessed by measuring plasma alanine and aspartate aminotransferase activities, and by determining reduced and oxidized glutathione in plasma and bile. Hepatocyte function was quantitated by determining bile flow and liver ATP content. Ischemia-reperfusion resulted in severe hepatic injury involving a huge increase in alanine and aspartate aminotransferase activities, a drop in ATP content, and a decrease in bile flow. Plasma and bile reduced (GSH) and oxidized (GSSG) glutathione concentrations were inversely related: plasma levels increased when biliary levels decreased. This was associated with a decrease in animal survival (-34%). S-15176 pretreatment (1.25, 2.5, 5 or 10 mg kg(-1) day(-1)) improved the survival rate and limited tissue damages in a dose-dependent manner. The pretreatment also reduced the aminotransferase leakage from hepatocytes and the increase in plasma glutathione levels. In addition, normalization of the plasma GSSG/GSH ratio, a good index of an oxidative stress, was observed in groups treated with the higher dosage, suggesting that the antioxidant properties demonstrated for the compound in vitro (IC(50)=0.3 microM towards lipid peroxidation) could play a role in its protective effect. S-15176 pretreatment also protected the organ from the drop in ATP levels. At the higher dose, ATP content was maintained at a level almost 86% of the sham-operated group after 60 min of reperfusion. This was associated with a restoration of the biliary flow. These data suggest that S-15176 may be a useful drug in liver surgery to prevent ischemia-reperfusion injury.
Assuntos
Antioxidantes/farmacologia , Isquemia/tratamento farmacológico , Fígado/irrigação sanguínea , Piperazinas/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Trifosfato de Adenosina/metabolismo , Animais , Bile/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , RatosRESUMO
Cyclosporine A (CsA) is a known potent inhibitor of pro-oxidant-induced mitochondrial swelling. In the present study we show that CsA's effect is only transient when the liver mitochondrial swelling in induced by Ca2+ plus tert-butylhydroperoxide (t-BH). After an initial inhibition, swelling is worsened by CsA as evidenced by an extent of mitochondrial swelling that exceeds that of the control. Unlike CsA, trimetazidine (TMZ), an anti-ischemic drug decreases both the extent and the rate of the swelling with an IC50 value of 214 +/- 24 microM. Its inhibition effect on the initial swelling rate mimicks that of CsA but the mechanism may be independent. During long-term swelling. TMZ counteracts the worsening effect of CsA. The inhibition of swelling induced by TMZ is assessed by the fact that TMZ significantly increases the EC50 of Ca(2+)-induced mitochondrial swelling (46.6 +/- 6.0 to 85 +/- 10 microM, P < 0.01), without affecting its cooperativity. Apparently, TMZ seems to behave like trifluoperazine (TFP), a phospholipase A2 inhibitor that, under our experimental conditions, inhibits the mitochondrial swelling induced by Ca2+ and t-BH with an IC50 value of 25 +/- 10 microM. Both drugs are able to protect mitochondria from both phases (early and late) of the swelling, especially the late, which is enhanced in the presence of CsA. TFP and other phospholipase A2 inhibitors were able to displace [3H]TMZ from its mitochondrial binding sites whereas CsA was ineffective. We suggest that TMZ, like TFP, inhibits the CsA insensitive mechanism involved in the swelling process which is responsible for the worsening effect observed in the presence of CsA when the swelling is generated by Ca2+ and t-BH.
Assuntos
Ciclosporina/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Trimetazidina/farmacologia , Animais , Cálcio/farmacologia , Masculino , Mitocôndrias Hepáticas/fisiologia , Ratos , Ratos Wistar , Trifluoperazina/farmacologiaRESUMO
Although the role of Ca(2+) in liver transplantation injury has been the object of several studies, direct evidence for alterations in intracellular Ca(2+) homeostasis after cold preservation-warm reoxygenation (CP/WR) has never been presented. We thus investigated the effects of CP/WR on steady-state Ca(2+) and responses to a Ca(2+)-mobilizing agonist. Isolated rat hepatocytes were suspended in University of Wisconsin solution, stored at 4 degrees C for 0, 24, and 48 h, and reoxygenated at 37 degrees C for 1 h. Cytosolic Ca(2+) was measured in single cells by digitized fluorescence videomicroscopy. CP/WR caused a significant increase in steady-state cytosolic Ca(2+), which was inversely proportional to cell viability. Pretreatment of hepatocytes with an agent that protects mitochondrial function attenuated the increase in steady-state cytosolic Ca(2+) and improved hepatocyte viability. Ca(2+) responses to the purinergic agonist ATP also increased significantly as a function of cold storage time. This increase was related to an increase in the size of inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores and subsequent capacitative Ca(2+) entry. Thus CP/WR significantly perturbs steady-state hepatocellular Ca(2+) and responses to Ca(2+)-mobilizing agonists, which may contribute to hepatocyte metabolic dysfunction observed after CP/WR.
Assuntos
Cálcio/metabolismo , Temperatura Baixa , Hepatócitos/metabolismo , Oxigênio/metabolismo , Temperatura , 3-Iodobenzilguanidina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citoproteção , Relação Dose-Resposta a Droga , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/farmacologia , Líquido Intracelular/metabolismo , Masculino , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxigênio/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Ischaemia-reperfusion induces structural and functional damage to hepatocytes. The purpose of this study was to evaluate the protective effect of trimetazidine, an anti- ischaemic drug, in a rat liver model of ischaemia-reperfusion. Male Wistar rats were divided into groups pretreated with different doses of trimetazidine (1, 5, 10 or 20 mg kg-1 day-1) or saline for 7 days. Liver ischaemia was induced for 120 min and blood reflow was subsequently restored for 30, 60, 90 or 120 min. The activities of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) as well as the bile flow and the liver ATP content were determined. Ischaemia-reperfusion induced major alterations of hepatic functions involving increases of ASAT and ALAT activities, a drop of ATP content and a sharp decrease in bile flow. Trimetazidine pretreatment reduced the liver injury. Indeed, it lowered the increase in ALAT and ASAT activities observed immediately after reperfusion and maintained higher concentrations of hepatic ATP. Simultaneously, bile flow was increased. These effects were dose-dependent and 5 mg kg-1 day-1 seemed to be the lowest effective dose. In this experimental model trimetazidine pretreatment reduced the liver damage induced by ischaemia-reperfusion. Our data suggest that trimetazidine may be a useful drug in liver surgery to prevent ischaemia-reperfusion injury.
Assuntos
Hepatopatias/prevenção & controle , Substâncias Protetoras/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Trimetazidina/uso terapêutico , Animais , Masculino , Ratos , Ratos WistarRESUMO
Recent studies suggest a crucial role played by mitochondria in the pathogenesis of ischemia-reperfusion injury. This study was conducted to clarify the role of trimetazidine, a cellular anti-ischemic agent, on mitochondria isolated from rat liver subjected to 120-min normothermic ischemia followed by 30-min reperfusion. Rats were divided into groups, pretreated with different doses of trimetazidine (5, 10 and 20 mg/kg/day) or saline and subjected to the ischemia-reperfusion process; another group served as the sham-operated controls. Alanine aminotransferase and aspartate aminotransferase activities and hepatocyte ATP content, bile flow and mitochondrial functions were assessed. Ischemia-reperfusion caused membrane leakage from hepatocytes and a decrease in ATP content and in bile flow. These effects were well correlated with alterations in mitochondrial function, namely, decrease in ATP synthesis, NAD(P)H level and mitochondrial membrane potential and generation of mitochondrial permeability transition. The pretreatment of rats with trimetazidine prevented these ischemia-reperfusion deleterious effects at both the cellular and mitochondrial level in a dose-dependent manner. It is concluded that trimetazidine at an optimal dosage of 10 mg/kg/day protects mitochondria against the deleterious effects of ischemia-reperfusion. This protective effect appears to be the key factor through which this drug exerts its cytoprotective activity.