Swelling Of Rat Liver Mitochondria Research Articles

Physiological changes of mitochondria under the diabetes condition and pharmacological approaches to eliminate them

The state of lipid peroxidation (LPO), respiration and oxidative phosphorylation, mitochondrial permeability transition pore (mPTP), and the antiradical activity of rat liver mitochondria in the streptozotocin (STZ)-induced diabetes condition were studied with the consideration of the ways of correcting detected membrane damages using gossitan isolated from the cotton plant Gossypium hirsutum L. It was shown that the rate of respiration of liver mitochondria in states V3 and V4 increases during STZ-induced diabetes, which significantly reduces the respiratory control (RC) and ADP/O coefficients in comparison with the control. The findings suggest that the uncoupling of respiration and oxidative phosphorylation takes place during STZ-induced diabetes. It was shown that in the STZ-induced diabetes condition, the rate of swelling of rat liver mitochondria is higher than that of the healthy ones; this means that mPTP of rat liver mitochondria is in the open state. Gossitan recovers mPTP to the normal condition, thereby removing the STZ effect on mitochondria. Gossitan (a personal dose is 10 mg/kg of body weight, during 8 days) eliminates the detected functional disorders of rat liver mitochondria, probably due to its antioxidant properties.

In vitro effects of 2-methyl-3-propylbutane-1,4-diol purified from Alstonia boonei on erythrocyte membrane stabilization and mitochondrial membrane permeabilization.

A recent review on the ethnomedicinal, chemical, pharmacological, and toxicological properties of Alstonia boonei revealed the plant's potential in the treatment and management of a range of diseases. However, most of these pharmacological effects are only traceable to the crude form of the plant extract and not specific natural products. Phytochemical investigation of the methanol fraction of the methanol extract of the stem-bark of Alstonia boonei led to the isolation and identification of 2-methyl-3-propylbutane-1,4-diol. The structures were elucidated by the application of 1D-, and 2D-NMR spectroscopic analyses and by comparison with literature data. In this study, the membrane stabilizing activity, mitochondrial membrane permeability transition pore opening, cytochrome c release, mitochondrial ATPase activity, and prevention of mitochondrial lipid peroxidation activity of 2-methyl-3-propylbutane-1,4-diol (MPBD) isolated from A. boonei were determined. The results showed that MPBD significantly (p < .05) prevented peroxidation of mitochondrial membrane lipids and hemolysis using both the heat-induced and hypotonic solution-induced membrane stabilization assays. On the contrary, the compound caused large amplitude swelling of rat liver mitochondria in the absence of calcium, significant (p < .05) cytochrome c release and enhancement of mitochondrial ATPase activity in vitro. Our findings suggest that MPBD showed characteristic biological properties useful in modulating cell death.

PHYSIOLOGICAL ALTERATIONS OF MITOCHONDRIA UNDER DIABETES CONDITION AND ITS CORRECTION BY POLYPHENOL GOSSITAN

The state of lipid peroxidation (LPO), respiration and oxidative phosphorylation, mitochondrial permeability transition pore (mPTP) and antiradical activity of rat liver mitochondria in streptozotocin (STZ)-induced diabetes condition were studied, considered the ways of correction of detected membrane damages with the use of gossitan, isolated from cotton plant Gossypium hirsutum L. It was shown that the rate of respiration of liver mitochondria in states V3 and V4 increases during STZ-induced diabetes, which significantly reduces the respiratory control (RC) and ADP/O coefficients in comparison with the control. The findings suggest that the uncoupling of respiration and oxidative phosphorylation takes place during STZ-induced diabetes. It was shown that in the conditions of STZ-induced diabetes, the rate of swelling of rat liver mitochondria is higher than of the healthy ones; this means that mPTP of rat liver mitochondria is in the open state. Gossitan recovers mPTP to the normal condition, thereby removing the effect of STZ on mitochondria. Gossitan (per os dose is 10 mg/kg of body weight, during 8 days) eliminates the detected functional disorders of rat liver mitochondria, probably due to its antioxidant properties.

Age-Dependent Changes in the Function of Mitochondrial Membrane Permeability Transition Pore in Rat Liver Mitochondria

Mitochondria play an important role in the cell aging process. Changes in calcium homeostasis and/or increased reactive oxygen species (ROS) production lead to the opening of mitochondrial permeability transition pore (MPTP), depolarization of the inner mitochondrial membrane, and decrease of ATP production. Our work aimed to monitor age-related changes in the Ca2+ ion effect on MPTP and the ability of isolated rat liver mitochondria to accumulate calcium. The mitochondrial calcium retention capacity (CRC) was found to be significantly affected by the age of rats. Measurement of CRC values of the rat liver mitochondria showed two periods when 3 to17-week old rats were tested. 3-week and 17-week old rats showed lower CRC values than 7-week old animals. Similar changes were observed while testing calcium-induced swelling of rat liver mitochondria. These findings indicate that the mitochondrial energy production system is more resistant to calcium-induced MPTP opening accompanied by the damaging effect of ROS in adult rats than in young and aged animals.

Age-Dependent Changes in the Function of Mitochondrial Membrane Permeability Transition Pore in Rat Liver Mitochondria.

Mitochondria play an important role in the cell aging process. Changes in calcium homeostasis and/or increased reactive oxygen species (ROS) production lead to the opening of mitochondrial permeability transition pore (MPTP), depolarization of the inner mitochondrial membrane, and decrease of ATP production. Our work aimed to monitor age-related changes in the Ca2+ ion effect on MPTP and the ability of isolated rat liver mitochondria to accumulate calcium. The mitochondrial calcium retention capacity (CRC) was found to be significantly affected by the age of rats. Measurement of CRC values of the rat liver mitochondria showed two periods when 3 to 17-week old rats were tested. 3-week and 17-week old rats showed lower CRC values than 7-week old animals. Similar changes were observed while testing calcium-induced swelling of rat liver mitochondria. These findings indicate that the mitochondrial energy production system is more resistant to calcium-induced MPTP opening accompanied by the damaging effect of ROS in adult rats than in young and aged animals.

Influence of Microbial Metabolites on the Nonspecific Permeability of Mitochondrial Membranes under Conditions of Acidosis and Loading with Calcium and Iron Ions.

Mitochondrial dysfunction is currently considered one of the main causes of multiple organ failure in chronic inflammation and sepsis. The participation of microbial metabolites in disorders of bioenergetic processes in mitochondria has been revealed, but their influence on the mitochondrial membrane permeability has not yet been studied. We tested the influence of various groups of microbial metabolites, including indolic and phenolic acids, trimethylamine-N-oxide (TMAO) and acetyl phosphate (AcP), on the nonspecific permeability of mitochondrial membranes under conditions of acidosis, imbalance of calcium ions and excess free iron, which are inherent in sepsis. Changes in the parameters of the calcium-induced opening of the mitochondrial permeability transition pore (MPTP) and iron-activated swelling of rat liver mitochondria were evaluated. The most active metabolites were indole-3-carboxylic acid (ICA) and benzoic acid (BA), which activated MPTP opening and swelling under all conditions. AcP showed the opposite effect on the induction of MPTP opening, increasing the threshold concentration of calcium by 1.5 times, while TMAO activated swelling only under acidification. All the redox-dependent effects of metabolites were suppressed by the lipid radical scavenger butyl-hydroxytoluene (BHT), which indicates the participation of these microbial metabolites in the activation of membrane lipid peroxidation. Thus, microbial metabolites can directly affect the nonspecific permeability of mitochondrial membranes, if conditions of acidosis, an imbalance of calcium ions and an excess of free iron are created in the pathological state.

Dodecyltriphenylphosphonium As an Inducer of Potassium-Dependent Permeability in Rat Liver Mitochondria

Dodecyltriphenylphosphonium (C12TPP) is a membrane-bound penetrating cation with a delocalized charge widely used for creating and delivering of antioxidants, spin traps, and other chemical probes into mitochondria. The ability of C12TPP to induce permeabilization of the inner membrane of rat liver mitochondria at micromolar concentrations was investigated. It was found that C12TPP in a dose-dependent manner (10–30 µM) caused swelling of rat liver mitochondria in the incubation medium, the osmolarity of which was maintained with KCl. At the same time, C12TPP induced the reduction of the volume of rat liver mitochondria in the isotonic sucrose medium. It was shown that mitochondrial swelling induced by C12TPP was not inhibited by cyclosporin A, but it was suppressed by glibenclamide and ATP, the inhibitors of the ATP-sensitive potassium channel. It was also found that the C12TPP-induced mitochondrial swelling was inhibited by free saturated fatty acids. In turn, C12TPP prevented mitochondrial swelling induced by palmitic acid and Ca2+, which was insensitive to cyclosporin A. A possible mechanism of the C12TPP-induced permeabilization of the inner mitochondrial membrane is discussed.

Effect of Dequalinium on Respiration and the Inner Membrane Permeability of Rat Liver Mitochondria

The effect of the lipophilic penetrating cation dequalinium on rat liver mitochondria was studied. It was found that dequalinium dose-dependently inhibits the respiration rate of rat liver mitochondria in ADP-stimulated (V3) and DNP-stimulated (uncoupled) states. This can be due to the fact that dequalinium is a potent inhibitor of complex III of the mitochondrial respiratory chain. It was shown that dequalinium induces a high-amplitude swelling of rat liver mitochondria. The dequalinium-induced swelling of the organelles depends on the presence of inorganic phosphate in the incubation medium: in the absence of phosphate or in the presence of the phosphate carrier inhibitor mersalyl in the phosphate-containing medium, no swelling of the mitochondria was observed. At low concentrations of dequalinium (≤10 μM), this swelling is inhibited by cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. At the same time, at high concentrations of dequalinium (>10 μM), cyclosporin A becomes ineffective. It was found that in the presence of dequalinium the rate of the H2O2 production increased in rat liver mitochondria. Possible mechanisms of toxic effect of dequalinium chloride are discussed.

Effect of the CRAC Peptide, VLNYYVW, on mPTP Opening in Rat Brain and Liver Mitochondria.

The translocator protein (TSPO; 18 kDa) is a high-affinity cholesterol-binding protein located in the outer membrane of mitochondria. A domain in the C-terminus of TSPO was characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide may participate in the regulation of mitochondrial membrane permeability. Herein, we report the effect of the synthetic CRAC peptide, VLNYYVW, on mitochondrial permeability transition pore (mPTP) opening. It was found that the CRAC peptide alone prevents the mPTP from opening, as well as the release of apoptotic factors (cytochrome c, AIF, and EndoG) in rat brain mitochondria (RBM). Co-incubation of CRAC, together with the TSPO drug ligand, PK 11195, resulted in the acceleration of mPTP opening and in the increase of apoptotic factor release. VLNYYVW did not induce swelling in rat liver mitochondria (RLM). 3,17,19-androsten-5-triol (19-Atriol; an inhibitor of the cholesterol-binding activity of the CRAC peptide) alone and in combination with the peptide was able to stimulate RLM swelling, which was Ca2+- and CsA-sensitive. Additionally, a combination of 19-Atriol with 100 nM PK 11195 or with 100 µM PK 11195 displayed the opposite effect: namely, the addition of 19-Atriol with 100 µM PK 11195 in a suspension of RLM suppressed the Ca2+-induced swelling of RLM by 40%, while the presence of 100 nM PK 11195 with 19-Atriol enhanced the swelling of RLM by 60%. Taken together, these data suggest the participation of the TSPO’s CRAC domain in the regulation of permeability transition.

Study on Biological Effects of La(3+) on Rat Liver Mitochondria by Microcalorimetric and Spectroscopic Methods.

The effects of lanthanum on heat production of mitochondria isolated from Wistar rat liver were investigated with microcalorimetry; simultaneously, the effects on mitochondrial swelling and membrane potential (Δψ) were determined by spectroscopic methods. La(3+) showed only inhibitory action on mitochondrial energy turnover with IC50 being 55.8μmolL(-1). In the spectroscopic experiments, La(3+), like Ca(2+), induced rat liver mitochondrial swelling and decreased membrane potential (Δψ), which was inhibited by the specific permeability transition inhibitor, cyclosporine A (CsA). The induction ability of La(3+) was stronger than that of Ca(2+). These results demonstrated that La(3+) had some biotoxicity effect on mitochondria; the effects of La(3+) and Ca(2+) on rat liver mitochondrial membrane permeability transition (MPT) are different, and La represents toxic action rather than Ca analogy.

Dimebon Inhibits Calcium-Induced Swelling of Rat Brain Mitochondria But Does Not Alter Calcium Retention or Cytochrome C Release

Dimebon was originally introduced as an antihistamine and subsequently investigated as a possible therapeutic for a variety of disorders, including Alzheimer's disease. One putative mechanism underlying the neuroprotective properties of Dimebon is inhibition of mitochondrial permeability transition, based on the observation that Dimebon inhibited the swelling of rat liver mitochondria induced by calcium and other agents that induce permeability transition. Because liver and brain mitochondria differ substantially in their properties and response to conditions associated with opening of the permeability transition pore, we sought to determine whether Dimebon inhibited permeability transition in brain mitochondria. Dimebon reduced calcium-induced mitochondrial swelling but did not enhance the calcium retention capacity or impair calcium-induced cytochrome C release from non-synaptic mitochondria isolated from rat brain cerebral cortex. These findings indicate that Dimebon does not inhibit mitochondrial permeability transition, induced by excessive calcium uptake, in brain mitochondria.

Physiological aspects of the mitochondrial cyclosporin A-insensitive palmitate/Ca2+-induced pore: tissue specificity, age profile and dependence on the animal’s adaptation to hypoxia

Earlier we found that being added to rat liver mitochondria, palmitic acid (Pal) plus Ca(2+) opened a cyclosporin A-insensitive pore, which remained open for a short time. Apparently, this pore is involved in the Pal-induced apoptosis and may also take part in the mitochondrial Ca(2+) recycling as a Ca(2+) efflux system (Belosludtsev et al. J Bioenerg Biomembr 38:113-120, 2006; Mironova et al. J. Bioenerg. Biomembr. 39:167-174, 2007). In this paper, we continue studying physiological and regulatory aspects of the pore. The following observations have been made. (1) Cardiolipin has been found to facilitate the Ca(2+)-induced formation of pores in the Pal-containing liposomal membranes. (2) The opening of Pal/Ca(2+)-induced pore is accompanied by the release of apoptosis-induced factor (AIF) from mitochondria. (3) The rate of Pal/Ca(2+)-induced swelling of rat liver mitochondria increases substantially with the age of animals. (4) Although the Pal/Ca(2+)-induced pore opens both in the liver and heart mitochondria, the latter require higher Pal concentrations for the pore to open. (5) The pore opening depends on the resistance of animals to hypoxia: in the highly resistant to hypoxia rats, the mitochondrial Pal/Ca(2+)-induced pore opens easier than in the low resistant animals, this being opposite for the classical, cyclosporin A-sensitive MPT pore. The adaptation of the low resistant rats to oxygen deficiency increases the sensitivity of their mitochondria to PalCaP inductors. The paper also discusses a possible role of the mitochondrial Pal/Ca(2+)-induced pore in the protection of tissues against hypoxia.

Impairment of mitochondrial function by minocycline

There is an ongoing debate on the presence of beneficial effects of minocycline (MC), a tetracycline-like antibiotic, on the preservation of mitochondrial functions under conditions promoting mitochondria-mediated apoptosis. Here, we present a multiparameter study on the effects of MC on isolated rat liver mitochondria (RLM) suspended either in a KCl-based or in a sucrose-based medium. We found that the incubation medium used strongly affects the response of RLM to MC. In KCl-based medium, but not in sucrose-based medium, MC triggered mitochondrial swelling and cytochrome c release. MC-dependent swelling was associated with mitochondrial depolarization and a decrease in state 3 as well as uncoupled respiration. Swelling of RLM in KCl-based medium indicates that MC permeabilizes the inner mitochondrial membrane (IMM) to K(+) and Cl(-). This view is supported by our findings that MC-induced swelling in the KCl-based medium was partly suppressed by N,N'-dicyclohexylcarbodiimide (an inhibitor of IMM-linked K(+)-transport) and tributyltin (an inhibitor of the inner membrane anion channel) and that swelling was less pronounced when RLM were suspended in choline chloride-based medium. In addition, we observed a rapid MC-induced depletion of endogenous Mg(2+) from RLM, an event that is known to activate ion-conducting pathways within the IMM. Moreover, MC abolished the Ca(2+) retention capacity of RLM irrespective of the incubation medium used, most likely by triggering permeability transition. In summary, we found that MC at low micromolar concentrations impairs several energy-dependent functions of mitochondria in vitro.

The prevention of curcumin against rat liver mitochondrial swelling induced by tert-butylhydroperoxide

Liver diseases have been a medical problem which is difficult to manage. Some of the problems in the treatment of these diseases lie in the lack of reliable drug available. Curcumin, an active ingredient of the rhizomes of plant Curcuma has been investigated in the treatment of various disorders incuding liver diseases. The therapeutic effects of curcumin on liver diseases have been thought to be associated to its antioxidative properties. In the present study, we investigated the effects of curcumin on mitochondrial swelling in vitro induced by tert-butylhydroperoxide (t-BuOOH). Liver mitochondria were homogeneously isolated from Sprague-Dawley rats (the relative specific activity of succinate dehydrogenase was 35.73 ± 2.78). Addition of 90 µM of t-BuOOH caused a typical 2-phase swelling of the mitochondria. The pattern of swelling was influenced by various factors such as buffer composition, concentrations of t-BuOOH, amount of isolation buffer and mitochondrial proteins and incubation temperature.The swelling could be reduced by as much as 85 ± 3% by 2.50 µM of curcumin. At lower (1.25 µM) or higher (5.00 µM) concentrations, the protection against swelling by curcumin were less effective (respectively were 41 ± 3% and 77 ± 6%). Swelling might occur due to the opening of mitochondrial transition pore and could be an initial indication in the cascade process leading to cell death. The inhibition of t-BuOOH-induced mitochondrial swelling by curcumin might be because of the antioxidant effects of the compound. (Med J Indones 2006; 15:131-6) Keywords: mitochondria, swelling, tert-butylhydroperoxide, curcumin

Novel cyclophilin D inhibitors derived from quinoxaline exhibit highly inhibitory activity against rat mitochondrial swelling and Ca2+ uptake/ release.

Aim:To investigate methods for identifying specific cyclophilin D (CypD) inhibitors derived from quinoxaline, thus developing possible lead compounds to inhibit mitochondrial permeability transition (MPT) pore opening.Methods:Kinetic analysis of the CypD/inhibitor interaction was quantitatively performed by using surface plasmon resonance (SPR) and fluorescence titration (FT) techniques. IC50 values of these inhibitors were determined by PPIase inhibition activity assays.Results:All the equilibrium dissociation constants (KD) of the seven compounds binding to CypD were below 10 μmol/L. The IC50 values were all consistent with the SPR and FT results. Compounds GW2, 5, 6, and 7 had high inhibition activities against Ca2+-dependent rat liver mitochondrial swelling and Ca2+ uptake/release. Compound GW5 had binding selectivity for CypD over CypA.Conclusion:The agreement between the measured IC50 values and the results of SPR and FT suggests that these methods are appropriate and powerful methods for identifying CypD inhibitors. The compounds we screened using these methods (GW1-7) are reasonable CypD inhibitors. Its potent ability to inhibit mitochondrial swelling and the binding selectivity of GW5 indicates that GW5 could potentially be used for inhibiting MPT pore opening.

Mitochondria Permeabilization by a Novel Polycation Peptide BTM-P1

Bacillus thuringiensis subsp. medellin is known to produce the Cry11Bb protein of 94 kDa, which is toxic for mosquito larvae due to permeabilization of the plasma membrane of midgut epithelial cells. Earlier we found that a 2.8-kDa novel peptide BTM-P1, which was artificially synthesized taking into account the primary structure of Cry11Bb endotoxin, is active against several species of bacteria. In this work we show that BTM-P1 induces cyclosporin A-insensitive swelling of rat liver mitochondria in various salt solutions but not in the sucrose medium. Inorganic phosphate and Ca(2+) significantly increased this effect of the peptide. The uncoupling action of BTM-P1 on oxidative phosphorylation was stronger in the potassium-containing media and correlated with a decrease of the inner membrane potential of mitochondria. In isotonic KNO(3), KCl, or NH(4)NO(3) media, a complete drop of the inner membrane potential was observed at 1-2 microg/ml of the peptide. The peptide-induced swelling was increased by energization of mitochondria in the potassium-containing media, but it was inhibited in the NaNO(3), NH(4)NO(3), and Tris-NO(3) media. All mitochondrial effects of the peptide were completely prevented by adding a single N-terminal tryptophan residue to the peptide sequence. We suggest a mechanism of membrane permeabilization that includes a transmembrane- and surface potential-dependent insertion of the polycation peptide into the lipid bilayer and its oligomerization leading to formation of ion channels and also to the mitochondrial permeability transition pore opening in a cyclosporin A-insensitive manner.

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+.

Activation of ion-conducting pathways in the inner mitochondrial membrane – an unrecognized activity of fatty acid?

The effect of non-esterified myristate (C14:0) or dodecyl sulfate was studied on passive swelling of rat liver mitochondria suspended in hypotonic alkaline KCl medium in the absence of the potassium ionophore valinomycin. Both compounds rapidly initiated large-amplitude swelling. However, they failed to initiate swelling when the mitochondria were suspended in hypotonic alkaline sucrose medium. In contrast to myristate or dodecyl sulfate, the non-ionic detergent Triton X-100 initiated swelling of mitochondria in both of the media. The following findings indicate that the inner mitochondrial membrane (IMM) is permeabilized by myristate to K + and Cl − in a specific manner. (i) Swelling initiated by myristate did not respond to cyclosporin A, (ii) the protonophoric uncoupler FCCP was unable to mimic the myristate effect on swelling, and (iii) myristate-induced Cl −-permeation (measured with KCl medium plus valinomycin) was inhibited by N, N′-dicyclohexylcarbodiimide, quinine or ATP. Myristate- or dodecyl sulfate-initiated swelling was paralleled by the lowering of endogenous Mg 2+ content. Both effects, stimulation of swelling and depletion of endogenous Mg 2+ are correlated with each other. Similar effects have been reported previously for the carboxylic divalent cation ionophore calcimycin (A23187). The A23187-induced swelling has identical inhibiting characteristics on Cl −-permeation with respect to N, N′-dicyclohexylcarbodiimide, quinine and ATP as the myristate-stimulated swelling. Therefore, we conclude that non-esterified fatty acids increase the permeability of mitochondria to K + and Cl − at alkaline pH by activating Mg 2+-dependent ion-conducting pathways in IMM.

Derivatives of 1,4-dihydropyridines as modulators of ascorbate-induced lipid peroxidation and high-amplitude swelling of mitochondria, caused by ascorbate, sodium linoleate and sodium pyrophosphate

A group of 26 2,6-dimethyl-3,5-disubstituted and 2,6-dimethyl-3,4, 5-trisubstituted-1,4-dihydropyridines (1,4-H(2)Py=1,4-DHPs) and five related pyridines were studied as inhibitors of rat liver mitochondrial swelling and O(2) uptake by ascorbic acid-dependent lipid peroxidation (LP) and as modulators of mitochondrial swelling induced by Na(+)-linoleate or Na(+)-pyrophosphate. 1,4-DHPs studied include 4-unsubstituted and 4-methyl- and 4-phenyl-substituted 3, 5-dialkoxycarbonylderivatives of 2,6-dimethyl-1,4-DHP with variations in alkoxy chain length and composition, 4-unsubstituted and 4-methyl-, 4-aryl- and 4-pyridyl-substituted 3, 5-dianilidocarbonylderivatives, and a structurally related group of 3,5-dipyridylamidocarbonylderivatives. Many 1,4-DHPs possess marked antioxidant (AO) and membrane stabilizing activity, expressed as the mitochondrial swelling (deltaA(520)/t) and/or O(2) uptake rate decrease (V(0)/V) as well as prolongation of the induction period (tau/tau(0)) of mitochondrial swelling and/or O(2) uptake at ascorbic acid-dependent LP of rat liver mitochondria. 4-Unsubstituted 3,5-dialkoxycarbonyl-2,6-dimethyl-1,4-DHPs, as well as 4-unsubstituted or those possessing lipophylic 4-aryl- groups 3, 5-diamido-2,6-dimethyl-1,4-DHPs, reveal marked AO and membrane stabilizing properties. Oxidized (heteroaromatized) derivatives have minimal activity. Perhaps 1,4-DHPs preferably act as antioxidants on stages of initiation and prolongation of LP chain reactions at low concentrations: IC(50) (when V(0)/V or tau/tau(0)=2) are 0.1 microM to 100 microM. At 100 microM 3,5-di-p-hydroxyphenoxycarbonyl- and 3, 5-di-p-tolyloxycarbonyl-2,6-dimethyl-1,4-DHPs, as well as 3, 5-diethoxycarbonyl-2,6-dimethylpyridine (oxidized form of Hantzsch ester) and 3,5-diamyloxycarbonyl-2,6-dimethylpyridine, alter the mitochondrial swelling rate in the presence of natural protonophore Na(+)-linoleate (0.063 mM and 0.125 mM). 3,5-Di-n-butyloxycarbonyl-2, 6-dimethyl-1,4-DHP at 100 microM completely stops mitochondrial swelling in the presence of 0.8 mM Na(+)-pyrophosphate. In the presence of many of the 1,4-DHPs, the lipid peroxidation process was inhibited. However, the swelling process could be prolonged, promoted, accelerated or inhibited-depending on 1,4-DHPs structure, concentration, the type of initiators of the swelling process and the medium composition.

Zinc as an Inducer of the Membrane Permeability Transition in Rat Liver Mitochondria

It is shown that 2–10 μM Zn2+induces swelling of rat liver mitochondria incubated in a buffered sucrose medium either with valinomycin or with FCCP, Ca2+, ionophore A23187, oligomycin, and nigericin. This swelling was associated with the release of GSH from mitochondria. Both processes were sensitive to known inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A, and Mg2+. Mitochondrial swelling induced by Zn2+was also inhibited by rotenone, antymycin A,N-ethylmaleimide, butylhydroxytoluene, and spermine, whereas it was stimulated bytert-butyl hydroperoxide, diamide, and monobromobimane. It did not require the addition of phosphate. The same sensitivity to pH of the mitochondrial swelling induced by Zn2+and by phenylarsine oxide suggests the same site of the interaction, namely, thiol groups. The ability of Zn2+to induce mitochondrial swelling gradually decreased along with its increasing concentration above 10 μM. It is concluded that micromolar Zn2+induces the MPT presumably by the interaction with cysteinyl residues. This process is independent of the mitochondrial membrane potential.