Quaternary ammonium surfactant structure determines selective toxicity towards bacteria: mechanisms of action and clinical implications in antibacterial prophylaxis.

OBJECTIVES: Broad-spectrum antimicrobial activity of quaternary ammonium surfactants (QAS) makes them attractive and cheap topical prophylactic options for sexually transmitted infections and perinatal vertically transmitted urogenital infections. Although attributed to their high affinity for biological membranes, the mechanisms behind QAS microbicidal activity are not fully understood. We evaluated how QAS structure affects antimicrobial activity and whether this can be exploited for use in prophylaxis of bacterial infections. METHODS: Acute toxicity of QAS to in vitro models of human epithelial cells and bacteria were compared to identify selective and potent bactericidal agents. Bacterial cell viability, membrane integrity, cell cycle and metabolism were evaluated to establish the mechanisms involved in selective toxicity of QAS. RESULTS: QAS toxicity normalized relative to surfactant critical micelle concentration showed n-dodecylpyridinium bromide (C12PB) to be the most effective, with a therapeutic index of ∼10 for an MDR strain of Escherichia coli and >20 for Neisseria gonorrhoeae after 1 h of exposure. Three modes of QAS antibacterial action were identified: impairment of bacterial energetics and cell division at low concentrations; membrane permeabilization and electron transport inhibition at intermediate doses; and disruption of bacterial membranes and cell lysis at concentrations close to the critical micelle concentration. In contrast, toxicity to mammalian cells occurs at higher concentrations and, as we previously reported, results primarily from mitochondrial dysfunction and apoptotic cell death. CONCLUSIONS: Our data show that short chain (C12) n-alkyl pyridinium bromides have a sufficiently large therapeutic window to be good microbicide candidates.

Toxicity of the herbicide linuron as assessed by bacterial and mitochondrial model systems.

Linuron is one of the most intensively used herbicides with predictable effects on the environment and non-target organisms. In the present study, two in vitro biological models (a Bacillus sp. and rat liver mitochondria) were used to evaluate linuron toxicity at a cell/subcellular level. Linuron inhibited bacterial growth and NADH-supported respiration, similar IC50 values being estimated for both toxic responses (74 and 98 µM, respectively). At concentrations up to 120 µM, linuron perturbed the respiration and phosphorylation efficiency of rat liver mitochondria, reflected by an increase of state 4 respiration and a decrease of the transmembrane potential, state 3 and FCCP-uncoupled respiration, when malate/glutamate or succinate were used as respiratory substrates. Consequently, a decrease of the respiratory control and ADP/O ratio was observed. This study suggests that linuron membrane interactions with adverse repercussions in the activity of membrane enzymatic complexes, such as those which constitute the prokaryotic and mitochondrial respiratory systems, may underlie the toxic effects exerted by that herbicide on non-target organisms. Moreover, this work contributes to the establishment of our bacterial model system as a good tool for chemical toxicity screening.

Mitochondrial dysfunction is the focus of quaternary ammonium surfactant toxicity to mammalian epithelial cells.

Surfactants have long been known to have microbicidal action and have been extensively used as antiseptics and disinfectants for a variety of general hygiene and clinical purposes. Among surfactants, quaternary ammonium compounds (QAC) are known to be the most useful antiseptics and disinfectants. However, our previous toxicological studies showed that QAC are also the most toxic surfactants for mammalian cells. An understanding of the mechanisms that underlie QAC toxicity is a crucial first step in their rational use and in the design and development of more effective and safer molecules. We show that QAC-induced toxicity is mediated primarily through mitochondrial dysfunction in mammalian columnar epithelial cell cultures in vitro. Toxic effects begin at sublethal concentrations and are characterized by mitochondrial fragmentation accompanied by decreased cellular energy charge. At very low concentrations, several QAC act on mitochondrial bioenergetics through a common mechanism of action, primarily by inhibiting mitochondrial respiration initiated at complex I and, to a lesser extent, by slowing down coupled ADP phosphorylation. The result is a reduction of cellular energy charge which, when reduced below 50% of its original value, induces apoptosis. The lethal effects are shown to be primarily a result of this process. At higher doses (closer to the critical micellar concentration), QAC induce the complete breakdown of cellular energy charge and necrotic cell death.

Mechanism of inhibition of mitochondrial ATP synthase by 17ß-estradiol.

17ß-estradiol (E2) is considered to modulate the ATP synthase activity through direct binding to the oligomycin sensitive-conferring protein. We have previously demonstrated that E2 increases the amplitude of depolarization associated with the addition of ADP to energized mitochondria (i.e., to initiate a phosphorylative cycle) suggesting a direct action on the phosphorylative system of mitochondria. The purpose of the present study was to investigate the underlying mechanisms responsible for this effect. We show here that E2 modulates the activity of mitochondrial ATP synthase by promoting the intrinsic uncoupling ("slipping") of the ATP synthase. E2 depressed RCR, ADP/O ratio and state 3 respiration, whereas state 4 respiration was increased and VFCCP (uncoupled respiration) remained unaltered. In contrast to the stimulatory effect on state 4 respiration, state 2 respiration and Volig were not affected by E2. The effect of E2 appeared to be directed towards ATP synthase, since glutamate/malate respiration, uncoupled from the electron transport chain, was unaffected by E2. Apparently, E2 allows a proton back-leak through the Fo component of ATP synthase. This action of E2 is dependent on the presence of ATP, is more pronounced at high membrane potentials, and it is reversed by oligomycin (a Fo-ATP synthase inhibitor) but not by resveratrol (a F1-ATP synthase inhibitor). Altogether, our data provide a mechanistic explanation for the effect of E2 at the level of mitochondrial ATP synthase.

Use of a calcium-sensitive electrode for studies on mitochondrial calcium transport.

Ca(2+)-sensitive electrode as a practical approach is used to follow Ca(2+) changes in the medium and particularly useful to study mitochondrial Ca(2+) uptake (or release); this method permits the continuous recording of Ca(2+) movements through the mitochondrial inner membrane. In this chapter, it is described how to prepare a Ca(2+)-sensitive electrode, and its application on mitochondrial studies with emphasis on the mitochondrial permeability transition.

Proton leak modulation in testicular mitochondria affects reactive oxygen species production and lipid peroxidation.

Mitochondrial proton leak can account for almost 20% of oxygen consumption and it is generally accepted that this process contributes to basal metabolism. In order to clarify the role of basal proton leak in testicular mitochondria, we performed a comparative study with kidney and liver mitochondrial fractions. Proton leak stimulated by linoleic acid and inhibited by guanosine diphosphate (GDP) was detected, in a manner that was correlated with protein levels for uncoupling protein 2 (UCP2) in the three fractions. Modulation of proton leak had an effect on reactive oxygen species production as well as on lipid peroxidation, and this effect was also tissue-dependent. However, a possible role for the adenine nucleotide transporter (ANT) in testicular mitochondria proton leak could not be excluded. The modulation of proton leak appears as a possible and attractive target to control oxidative stress with implications for male gametogenesis.

Comparative effects of 3,4-methylenedioxymethamphetamine and 4-methylthioamphetamine on rat liver mitochondrial function.

Ecstasy, which is used as a recreational drug, is a common street name for 3, 4-methylenedioxymethamphetamine (MDMA). Another drug of abuse chemically related, though less common than MDMA, is the amphetamine derivative 4-methylthioamphetamine (MTA). MDMA and MTA induce different systemic and organ-specific effects, including neurotoxicity, hyperthermia, nephrotoxicity, cardiotoxicity and hepatotoxicity. Therefore, it is clear that MDMA and MTA are responsible for inducing organ toxicity. The mechanisms underlying MDMA and MTA-induced hepatotoxicity are multifactorial, and therefore not completely understood. Recent findings indicate interference with cellular bioenergetics as an important toxicological feature of ecstasy. However, less is known about the involvement of mitochondria in MTA-induced hepatotoxicity. Thus, we compared the direct influence of MDMA and MTA on rat liver mitochondrial function [mitochondrial permeability transition (MPT), mitochondrial oxidative stress, and mitochondrial bioenergetics]. It was shown that MTA (from 0.025 up to 0.1mM) was more potent than MDMA (from 0.2 up to 0.5mM) in decreasing the sensitivity of rat liver mitochondria to MPT. However, higher concentrations of MTA (from 0.5 up to 2mM) were highly toxic to mitochondria. MTA simultaneously increased H(2)O(2) production in a monoamine oxidase (MAO)-dependent way, and uncoupled and inhibited mitochondrial respiration. In contrast, MDMA had only limited or no effects on these mitochondrial parameters. According to these results, it is possible to postulate that, depending on the concentration, MTA can potentially be more efficient in its effects on liver mitochondria than MDMA and, also, that its harmful effects may contribute to its hepatotoxicity.

Effects of 5-acetyl(carbamoyl)-6-methylsulfanyl-1,4-dihydropyridine-5-carbonitriles on rat liver mitochondrial function.

It is increasingly recognised that mitochondria are potential targets to pharmacological and toxicological actions of membrane-active agents, including some 1,4-dihydropyridines derivatives (DHPs). The 5-acetyl(carbamoyl)-6-methylsulfanyl-1,4-dihydropyridine-5-carbonitriles (OSI-1146, OSI-3701, OSI-3761, and OSI-9642) is a new group of DHPs with minor differences on the molecular structure. It has also been shown that OSI-1146 displays cardiovascular, antioxidant, and antiradical activities, whereas OSI-3701 and OSI-3761 display hepatoprotective activity. Due to their protective properties, this group of DHPs may be potentially useful for the treatment of several pathological processes, including those associated with oxidative stress. However, the cellular targets for their pharmacological actions have not been investigated. The presented study, using isolated rat liver mitochondria was designed to investigate if mitochondria are a cellular target for the pharmacological and/or toxicological actions of these new group of DHPs. We studied the direct influence of these DHPs on rat liver mitochondrial function [bioenergetics, membrane permeability transition (MPT), and oxidative stress]. It was shown that OSI-1146, OSI-3761, and OSI-9642, in the concentration range of up to 200 microM, interfered with mitochondrial bioenergetics by affecting complexes I and II of the mitochondrial respiratory chain, the ATPase activity, and mitochondrial inner membrane permeability to protons. However, the effects of OSI-1146 were higher than those of OSI-3761 and OSI-9642. The remaining compound, OSI-3701, had no effect on the bioenergetic parameters tested. All the compounds increased the susceptibility of mitochondria to MPT, but, OSI-3701, not affecting the bioenergetic parameters, was the most potent. Moreover, all the compounds protected mitochondria against lipid peroxidation induced by the oxidant pair ADP/Fe(2+), but OSI-1146 was also the most potent. In conclusion, our results indicate that mitochondria are the potential intracellular targets for both protective and toxicological actions of the DHP compounds studied, suggesting that the potential use of these compounds as therapeutic agents should carefully consider their toxic effects to mitochondria.

Toxicity assessment of the herbicide metolachlor comparative effects on bacterial and mitochondrial model systems.

Metolachlor is one of the most intensively used chloroacetamide herbicides. However, its effects on the environment and on non-target animals and humans as well as its interference at a cell/molecular level have not yet been fully elucidated. The aim of this study was: firstly, to evaluate the potential toxicity of metolachlor at a cell/subcellular level by using two in vitro biological model systems (a strain of Bacillus stearothermophilus and rat liver mitochondria); secondly, to evaluate the relative sensibility of these models to xenobiotics to reinforce their suitability for pollutant toxicity assessment. Our results show that metolachlor inhibits growth and impairs the respiratory activity of B.stearothermophilus at concentrations two to three orders of magnitude higher than those at which bacterial cells are affected by other pesticides. Also at concentrations significantly higher than those of other pesticides, metolachlor depressed the respiratory control ratio, membrane potential and respiration of rat liver mitochondria when malate/glutamate or succinate were used as respiratory substrates. Moreover, metolachlor impaired the respiratory activity of rat liver mitochondria in the same concentration range at which it inhibited bacterial respiratory system (0.4-5.0 micromol/mg of protein). In conclusion, the high concentration range at which metolachlor induces toxicity in vitro suggests that this compound is safer than other pesticides previously studied in our laboratory, using the same model systems. The good parallelism between metolachlor effects on both models and the toxicity data described in the literature, together with results obtained in our laboratory with other compounds, indicate the suitability of these systems to assess toxicity in vitro.

Sanguinarine cytotoxicity on mouse melanoma K1735-M2 cells--nuclear vs. mitochondrial effects.

Sanguinarine (SANG) is an alkaloid recognized to have anti-proliferative activity against various human tumour cell lines. No data is available on the susceptibility of advanced malignant melanoma to SANG, although this disease has a very poor prognosis if not detected in time due to the resistance to conventional chemotherapy. The present work was designed to study the nuclear and mitochondrial involvement in the pro-apoptotic effect of SANG in an invasive mouse melanoma cell line. The results obtained show that SANG is primarily accumulated by the cell nuclei, causing inhibition of cell proliferation and inducing cell death, as confirmed by an increase in sub-G1 peaks. At low concentrations, SANG induces mitochondrial depolarization in a sub-population of melanoma cells, which also generally displayed strong nuclear labelling of phosphorylated histone H2AX. Western blotting revealed an increase in p53, but not Bax protein, in both whole-cell extracts and in mitochondrial fractions. Isolated hepatic mitochondrial fractions revealed that SANG affects the mitochondrial respiratory chain, and has dual effects on mitochondrial calcium loading capacity. We suggest that SANG is able to induce apoptosis in metastatic melanoma cells. The knowledge of mitochondrial vs. nuclear effects of SANG is important in the development of this promising compound for clinical use against aggressive melanoma.

Mitochondria as the target for mildronate's protective effects in azidothymidine (AZT)-induced toxicity of isolated rat liver mitochondria.

Previously mildronate, an aza-butyrobetaine derivative, was shown to be a cytoprotective drug, through its mechanism of action of inhibition of carnitine palmitoyltransferase-1, thus protecting mitochondria from long-chain fatty acid accumulation and subsequent damage. Recently in an azidothymidine (AZT)-induced cardiotoxicity model in vivo (in mice), we have found mildronate's ability of protecting heart tissue from nuclear factor kappaB abnormal expression. Preliminary data also demonstrate cerebro- and hepatoprotecting properties of mildronate in AZT-toxicity models. We suggest that mildronate may target its action predominantly to mitochondria. The present study in isolated rat liver mitochondria was designed to clarify mitochondrial targets for mildronate by using AZT as a model compound. The aim of this study was to investigate: (1) whether mildronate may protect mitochondria from AZT-induced toxicity; and (2) which is the most critical target in mitochondrial processes that is responsible for mildronate's regulatory action. The results showed that mildronate protected mitochondria from AZT-induced damage predominantly at the level of complex I, mainly by reducing hydrogen peroxide generation. Significant protection of AZT-caused inhibition of uncoupled respiration, ADP to oxygen ratio, and transmembrane potential were also observed. Mildronate per se had no effect on the bioenergetics, oxidative stress, or permeability transition of rat liver mitochondria. Since mitochondrial complex I is the first enzyme of the respiratory electron transport chain and its damage is considered to be responsible for different mitochondrial diseases, we may account for mildronate's effectiveness in the prevention of pathologies associated with mitochondrial dysfunctions.

Comparative effects of three 1,4-dihydropyridine derivatives [OSI-1210, OSI-1211 (etaftoron), and OSI-3802] on rat liver mitochondrial bioenergetics and on the physical properties of membrane lipid bilayers: relevance to the length of the alkoxyl chain in positions 3 and 5 of the DHP ring.

The 1,4-dihydropyridines OSI-1210, OSI-1211 (etaftoron), and OSI-3802 are compounds with similar chemical structure. They differ by the length of the alkoxyl chain in positions 3 and 5 of the dihydropyridine (DHP) ring and by their pharmacological action characteristics. However, as far as we know, a clear relationship between the effects of these compounds and the length of the alkoxyl chain in positions 3 and 5 of the DHP has not been established. The goal of this study was to compare the influence of OSI-1210, OSI-1211 (etaftoron), and OSI-3802 on rat liver mitochondrial bioenergetics and on the physical properties of membrane lipid bilayers, correlating their actions with the length of the alkoxyl chain in positions 3 and 5 of the DHP ring. Using either glutamate/malate or succinate as respiratory substrates, all the compounds, in concentrations of up to 500 microM, depressed state 3 and uncoupled respiration, respiratory control (RCR) and ADP/O ratios, and phosphorylation rate, whereas state 4 respiration was stimulated. However, the stimulatory effect on state 4 induced by OSI-3802, the compound with the longest chain in positions 3 and 5 of the DHP ring, as well as its inhibitory effects on RCR and ADP/O ratios and phosphorylation rate were more pronounced than that induced by OSI-1210 and OSI-1211 (etaftoron), the compounds with the shortest and intermediate chains, respectively. Moreover, OSI-3802 maximized state 4 stimulation and minimized RCR and ADP/O ratios, and phosphorylation rate at a concentration of 100 microM, whereas low graduate effects were detected with OSI-1210 and OSI-1211 (etaftoron) for concentrations of up to 500 microM. At low concentrations (< or =30 microM), OSI-3802, like its analogue OSI-1212 (cerebrocrast), reduced the phase transition temperature, the cooperative unit size, and the enthalpy associated with the phase transition temperature of dimyristoylphosphatidylcholine (DMPC) membrane bilayers. A good correlation was established between the effects of 200 microM OSI-1210, OSI-1211 (etaftoron), and OSI-3802 on glutamate/malate- and succinate-dependent RCR of rat liver mitochondria and on the enthalpy change (Delta H) for the thermotropic profile of DMPC membrane bilayers at a 0.2 drug/DMPC molar ratio, indicating that the changes induced by these compounds on both mitochondrial membrane integrity and physical properties of DMPC membrane bilayers are strongly related to the length of the alkoxyl chain in positions 3 and 5 of the DHP ring. A putative relationship between membrane physical perturbation, bioenergetics impairment and the molecular characteristics of the compounds will be established as an approach to better understand their differentiated toxicological and pharmacological actions.

Sildenafil citrate concentrations not affecting oxidative phosphorylation depress H2O2 generation by rat heart mitochondria.

Sildenafil citrate (Viagra) is a potent and specific inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), which exhibits cardioprotective action against ischemia/reperfusion injury in intact and isolated heart. The mechanism of its cardioprotective action is not completely understood, but some results suggested that sildenafil exerts cardioprotection through the opening of mitochondrial ATP-sensitive K+ channels (mitoKATP). However, the impact of sildenafil citrate per se on isolated heart mitochondrial function is unknown. The goal of this study was to investigate the influence of the compound on mitochondrial function (bioenergetics, Ca2+-induced mitochondrial permeability transition, and hydrogen peroxide (H2O2) generation) in an attempt to correlate its known actions with effects on heart mitochondria. It was observed that sildenafil citrate concentrations of up to 50 muM did not significantly affect glutamate/malate-supported respiration in states 2, 3, 4, oligomycin-inhibited state 3, and uncoupled respiration. The respiratory control ratio (RCR), the ADP to oxygen ratio (ADP/O), the transmembrane potential (DeltaPsi), the phosphorylation rate, and the membrane permeability to H+, K+ and Ca2+ were not affected either. However, sildenafil citrate decreased H2O2 generation by mitochondria respiring glutamate/malate, and also decreased the formation of superoxide radical (O2 (*-) ) generated in a hypoxantine/xantine oxidase system. It was concluded that sildenafil citrate concentrations of up to 50 microM do not affect either rat heart mitochondrial bioenergetics or Ca2+-induced mitochondrial permeability transition, but it depresses H2O2 generation by acting as a superoxide dismutase (SOD)-mimetic. By preventing reactive oxygen species (ROS) generation, sildenafil citrate may preserve heart mitochondrial function.

Mitochondrially targeted effects of berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium] on K1735-M2 mouse melanoma cells: comparison with direct effects on isolated mitochondrial fractions.

Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium] is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment.

Inhibition of mitochondrial bioenergetics by carbaryl is only evident for higher concentrations -- Relevance for carbaryl toxicity mechanisms.

Although pesticides have been useful in agriculture pest control, there is a considerable risk for human health and damage to ecosystems. Carbaryl is a carbamate often taken as a safe insecticide, although data on metabolic activities is still scarce, viz. mitochondrial toxicity. Therefore, it is the goal of this work to assay the compound on isolated mitochondria, a biochemical model already used with other pesticides. Mitochondria isolated from the livers of Wistar rats were assayed for bioenergetic parameters, namely mitochondrial respiration, membrane potential, membrane integrity and enzyme activities. For higher concentrations, it was observed that carbaryl has a depressive effect on mitochondrial respiration and on the generation of mitochondrial membrane potential, but with preservation of membrane integrity. A locus between Complex II and III appears particularly affected and the mitochondrial phosphorylation system relatively insensitive. Therefore, carbaryl inhibits mitochondrial respiration without affecting the phosphorylation complex. Carbaryl is toxic for mitochondria, although at concentrations higher as compared with other insecticide compounds. Mitochondrial toxicity should be excluded as one of the primary causes for carbaryl immediate toxicity, as concluded from the range of concentrations where carbaryl shows effective mitochondrial toxicity.

Tetrandrine concentrations not affecting oxidative phosphorylation protect rat liver mitochondria from oxidative stress.

The effects of tetrandrine (6,6', 7,12-tetramethoxy-2, 2'-dimethyl-berbaman) on the mitochondrial function were assessed on oxidative stress, mitochondrial permeability transition (MPT), and bioenergetics of rat liver mitochondria. At concentrations lower than 100 nmol/mg protein, tetrandrine decreased the hydrogen peroxide formation, the extent of lipid peroxidation, the susceptibility to Ca(2+)-induced opening of MPT pore, and inhibited the inner membrane anion channel activity, not significantly affecting the mitochondrial bioenergetics. High tetrandrine concentrations (100-300 nmol/mg protein) stimulated succinate-dependent state 4 respiration, while some inhibition was observed for state 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations. The respiratory control ratio and the transmembrane potential were depressed but the adenosine diphosphate to oxygen (ADP/O) ratio was less affected. A slight increase of the inner mitochondrial membrane permeability to H(+) and K(+) by tetrandrine was also observed. It was concluded that low concentrations of tetrandrine afford protection against liver mitochondria injury promoted by oxidative-stress events, such as hydrogen peroxide production, lipid peroxidation, and induction of MPT. Conversely, high tetrandrine concentrations revealed toxicological effects expressed by interference with mitochondrial bioenergetics, as a consequence of some inner membrane permeability to H(+) and K(+) and inhibition of the electron flux in the respiratory chain. The direct immediate protective role of tetrandrine against mitochondrial oxidative stress may be relevant to clarify the mechanisms responsible for its multiple pharmacological actions.

Cerebrocrast promotes the cotransport of H+ and Cl- in rat liver mitochondria.

Considering that cerebrocrast stimulates oligomycin-inhibited state 3 respiration simultaneously with mitochondrial transmembrane potential (Deltapsi) dissipation, the mechanism underlying the uncoupler activity of cerebrocrast was assessed by its ability to permeabilize the mitochondrial inner membrane to H(+) or to K(+) or to cotransport anions with H(+). The partition coefficient of cerebrocrast in mitochondrial membrane and its ability to act as a membrane-active compound disturbing membrane lipid organization were also investigated. Cerebrocrast induced no permeabilization of mitochondrial inner membrane to H(+) or K(+), but it was able to transport H(+) in association with Cl(-). Cerebrocrast showed a strong incorporation into the mitochondrial membrane, with a partition coefficient (Kp(m/w)) of 2.7(+/-0.1)x10(5). Cerebrocrast also reduced, in a concentration dependent manner, the phase transition temperature, the cooperative unit size, and the enthalpy associated with the phase transition temperature of DMPC membrane bilayers. It was concluded that the uncoupler activity of cerebrocrast is due to its ability to promote the cotransport of H(+) with Cl(-) through the rat liver mitochondrial inner membrane, and that this cerebrocrast mechanism of action may be potentiated by alterations of membrane lipid organization and membrane lateral heterogeneity.

Glibenclamide interferes with mitochondrial bioenergetics by inducing changes on membrane ion permeability.

The interference of glibenclamide, an antidiabetic sulfonylurea, with mitochondrial bioenergetics was assessed on mitochondrial ion fluxes (H+, K+, and Cl-) by passive osmotic swelling of rat liver mitochondria in K-acetate, KNO3, and KCl media, by O2 consumption, and by mitochondrial transmembrane potential (Deltapsi). Glibenclamide did not permeabilize the inner mitochondrial membrane to H+, but induced permeabilization to Cl- by opening the inner mitochondrial anion channel (IMAC). Cl- influx induced by glibenclamide facilitates K+ entry into mitochondria, thus promoting a net Cl-/K+ cotransport, Deltapsi dissipation, and stimulation of state 4 respiration rate. It was concluded that glibenclamide interferes with mitochondrial bioenergetics of rat liver by permeabilizing the inner mitochondrial membrane to Cl- and promoting a net Cl-/K+ cotransport inside mitochondria, without significant changes on membrane permeabilization to H+.

Calcium-dependent mitochondrial permeability transition is augmented in the kidney of Goto-Kakizaki diabetic rat.

BACKGROUND: Renal disease associated with diabetes mellitus is a major problem among diabetic patients. The role of mitochondria in the pathogenesis of diabetes has received a large amount of attention in the last years, but many aspects of this subject are still poorly understood. In the present study, we studied the susceptibility of the mitochondrial permeability transition (MPT) on kidney mitochondria from the Goto-Kakizaki (GK) rat, an animal model featuring physiological and pathological alterations characteristic of type 2 diabetes. METHODS: Kidney mitochondria were isolated by differential centrifugations; mitochondrial electric transmembrane potential and calcium loading capacity were evaluated with a TPP+-selective electrode and with a calcium-sensitive fluorescent probe. Coenzyme Q9, Q10 and vitamin E were evaluated by high-performance liquid chromatography (HPLC). RESULTS: Kidney mitochondria from the diabetic animals had an increased susceptibility to the induction of the MPT by calcium. We observed a loss of calcium-loading capacity and a higher calcium-induced mitochondrial depolarization. Vitamin E and coenzyme Q9 were also increased in kidney mitochondria from GK rats. CONCLUSIONS: The results show an enhanced MPT activation in kidney mitochondria from GK rats, which lead us to suggest that this condition may be one major alteration triggered by chronic diabetes in kidney cells, ultimately leading to cell dysfunction.

Carvedilol inhibits the mitochondrial permeability transition by an antioxidant mechanism.

It was previously shown that carvedilol, a beta-adrenergic receptor antagonist with antioxidant properties, was able to inhibit the mitochondrial permeability transition (MPT). In the present work, the hypothesis was that the negative impact of carvedilol on the MPT was specifically the result of its antioxidant effect. For the current investigation, we used three different MPT inducers. MPT-associated events were tested to study the protective effect of both carvedilol and cyclosporin-A, the known MPT inhibitor. Carvedilol inhibited mitochondrial swelling with calcium plus phosphate and with calcium plus t-butylhydroperoxide, but not with calcium plus carboxyatractyloside. Carvedilol inhibited the oxidation of thiol groups with calcium plus phosphate (p < 0.01) and with calcium plus t-butylhydroperoxide (p < 0.05), but not with calcium plus carboxyatractyloside--in opposition to the full protection afforded by cyclosporin-A when using calcium and carboxyatractyloside. Our results showed that carvedilol was effective only when the MPT was triggered by a primary oxidative process. This finding implies that the antioxidant properties of carvedilol are crucial for the observed effects and reinforces the advantageous use of carvedilol in cardiac pathologies associated with enhanced cellular oxidative stress.