Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.     

Control of the mitochondrial inner membrane permeability by sulfhydryl groups

The effect of some thiol alkylating agents (N-substituted maleimide derivatives) on the permeability of the mitochondrial inner membrane was investigated. Several experimental approaches were used to study the modifications of the permeability properties. Alkylation of sulfhydryl groups led to an increase in the nonspecific permeability as judged by (i) the augmentation of the rate of osmotic shrinkage of mitochondria induced by polyethylene glycol, (ii) the sensitization of succinate dehydrogenase toward oxaloacetate, (iii) the enhancement of the oxidation rate of exogenous NADH, and (iv) the increase of the sucrose permeable space. The sulfhydryl groups involved in the maintenance of the selective permeability were shown to be located in the hydrophobic core of the membrane. Energization of mitochondria provoked an unmasking of these sulfhydryl groups. When magnesium ions were present in the incubation medium, N-substituted maleimide derivatives promoted gross modifications of the intramitochondrial ionic contents. Effluxes of endogenous calcium ions, inorganic phosphate, adenine nucleotides, and NAD(P)H were established. It was concluded that sulfhydryl groups probably play a crucial role in the maintenance of the membrane integrity and thus control the mitochondrial inner membrane permeability.     

Changes in the permeability of the inner mitochondrial membrane associated with plastid development

Summary Mitochondria isolated from greening etiolated laminae of Avena sativa L. show changes in the permeability of their inner membranes during chloroplast development similar to those described earlier for plastids. Oxalo-acetate, succinate and -keto-glutarate permeate most readily inner membranes of mitochondria isolated from laminae given 2 h illumination whilst glutamate and glycine show later and more general penetration into the matrix spaces of mitochondria from greening tissue. Aminolevulinic acid (ALA) by contrast does not readily enter Avena mitochondria especially those isolated from laminae illuminated for longer than 2 h.Abbreviations ALA amino-levulinic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane-sulphonic acid     

Protamine reversibly decreases paracellular cation permeability inNecturus gallbladder

Summary Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested inNecturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 g/ml=20 m), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage ( ^ms ) turned from slightly mucosapositive values to mucosa-negative values of approximately +1 to –5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 g/ml but a maximum response was achieved between 10 and 20 g/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5×10^–4 m) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.     

Prooxidants open both the mitochondrial permeability transition pore and a low-conductance channel in the inner mitochondrial membrane

Mitochondria can be induced by a variety of agents/conditions to undergo a permeability transition (MPT), which nonselectively increases the permeability of the inner membrane (i.m.) to small (<1500 Da) solutes. Prooxidants are generally considered to trigger the MPT, but some investigators suggest instead that prooxidants open a Ca(2+)-selective channel in the inner mitochondrial membrane and that the opening of this channel, when coupled with Ca(2+) cycling mediated by the Ca(2+) uniporter, leads ultimately to the observed increase in mitochondrial permeability [see, e.g., Schlegel et al. (1992) Biochem. J. 285, 65]. S. A. Novgorodov and T. I. Gudz [J. Bioenerg. Biomembr. (1996) 28, 139] propose that the i.m. contains a pore that, upon exposure to prooxidants, can open to two states, one of which conducts only H(+) and one of which is the classic MPT pore. Given the current interest in increased mitochondrial permeability as a factor in apoptotic cell death, it is important to determine whether i.m. permeability is regulated in one or multiple ways and, in the latter event, to characterize each regulatory mechanism in detail. This study examined the effects of the prooxidants diamide and t-butylhydroperoxide (t-BuOOH) on the permeability of isolated rat liver mitochondria. Under the experimental conditions used, t-BuOOH induced mitochondrial swelling only in the presence of exogenous Ca(2+) (>2 microM), whereas diamide was effective in its absence. In the absence of exogenous inorganic phosphate (P(i)), (1) both prooxidants caused a collapse of the membrane potential (DeltaPsi) that preceded the onset of mitochondrial swelling; (2) cyclosporin A eliminated the swelling induced by diamide and dramatically slowed that elicited by t-BuOOH, without altering prooxidant-induced depolarization; (3) collapse of DeltaPsi was associated with Ca(2+) efflux but not with efflux of glutathione; (4) neither Ca(2+) efflux nor DeltaPsi collapse was sensitive to ruthenium red; (5) collapse of DeltaPsi was accompanied by an increase in matrix pH; no stimulation of respiration was observed; (6) Sr(2+) was able to substitute for Ca(2+) in supporting t-BuOOH-induced i.m. depolarization, but not swelling; (7) in addition to being insensitive to CsA, the collapse of DeltaPsi was also resistant to trifluoperazine, spermine, and Mg(2+), all of which block the MPT; and (8) DeltaPsi was restored (and its collapse was inhibited) upon addition of dithiothreitol, ADP, ATP or EGTA. We suggest that these results indicate that prooxidants open two channels in the i.m.: the classic MPT and a low-conductance channel with clearly distinct properties. Opening of the low-conductance channel requires sulfhydryl group oxidation and the presence of a divalent cation; both Ca(2+) and Sr(2+) are effective. The channel permits the passage of cations, including Ca(2+), but not of protons. It is insensitive to inhibitors of the classic MPT.     

Effect of cyclosporine A and oligomycin on non-specific permeability of the inner mitochondrial membrane

The effect of oligomycin and cyclosporine A on the induction of non-specific permeability of the inner mitochondrial membrane by Ca²⁺ was under study. Both oligomycin and cyclosporine A were able to prevent the activation of non-specific permeability, but cyclosporine A was the only agent which could restore initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was shown not to be mediated through redistribution of Ca²⁺ between different mitochondrial subpopulations     

Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane

The mitochondrial permeability transition pore allows solutes with a m.w. 1500 to equilibrate across the inner membrane. A closed pore is favored by cyclosporin A acting at a high-affinity site, which may be the matrix space cylophilin isozyme. Early results obtained with cyclosporin A analogs and metabolites support this hypothesis. Inhibition by cyclosporin does not appear to require inhibition of calcineurin activity; however, it may relate to inhibition of cyclophilin peptide bond isomerase activity. The permeability transition pore is strongly regulated by both the membrane potential () and pH components of the mitochondrial protonmotive force. A voltage sensor which is influenced by the disulfide/sulhydryl state of vicinal sulfhydryls is proposed to render pore opening sensitive to . Early results indicate that this sensor is also responsive to membrane surface potential and/or to surface potential gradients. Histidine residues located on the matrix side of the inner membrane render the pore responsive to pH. The pore is also regulated by several ions and metabolites which act at sites that are interactive. There are many analogies between the systems which regulate the permeability transition pore and the NMDA receptor channel. These suggest structural similarities and that the permeability transition pore belongs to the family of ligand gated ion channels.     

Reducibility of cytochromeb in mitochondrial inner membrane

When mitochondrial inner membrane was disintegrated into Complex I-III, IV, and oligomycin-sensitive ATPase, about 50% of cytochromeb in Complex I-III was readily reduced with NADH, as judged by the appearance of a peak at 562 nm, while in whole mitochondria less than 25% of cytochromeb was reduced by succinate. On addition of antimycin to the substrate-reduced Complex I-III, cytochromeb was further reduced to 71% of the total, and the peak at 562 nm was red-shifted to 564 nm as in the case of dithionite reduction. These results indicate that the 562 nm and 564 nm peaks, at 29°C correspond, respectively, tob ₅₆₀ andb _562.5 at 77°K of Davis et al. [7] and to b _K and b _T of Chance et al. [2]. When Complex I-III and oligomycin-sensitive ATPase were reconstituted to form a membrane, about 60% of cytochromeb in Complex I-III was readily reduced with NADH. In this case the 562 nm peak was not red-shifted. However, the difference spectrum of NADH-reduced membraneminus that in the presence of deoxycholate showed a peak at 565 nm. A mirror image of the difference spectrum was obtained on addition of an uncoupler,m-chlorocarbonyl cyanide phenylhydrazone. This is characteristic for b _T. These results support the idea that the occurrence of spectral peaks of b _T and b _K is not due to two species but to single speciesAbbreviations OS-ATPase oligomycin sensitive ATPase - CCCP m-chlorocarbonyl cyanide phenylhydrazone - F₁ coupling factor one - OSCP oligomycinsensitivity-conferring protein     

Erythrocyte membrane sulfhydryl groups and cation permeability

Reaction of the slowly penetrating organic mercurial compound parachloromercuribenzene sulfonate (PCMBS) with intact erythrocytes has been characterized. Addition of concentrations of PCMBS which result in binding within the interior of the membrane of more than 1.9 × 10^?18 moles/cell produces alterations in Na⁺ and K⁺ permeability, but does not affect choline permeability. However, the increased cation permeability is observed only after a lag period of over two hours. After ten hours, a spontaneous slow “recovery” to normal rates of K⁺ leakage occurs at 25°C but not at 2°C. Subsequent to the effects on cation balance, increasing degrees of hemolysis occur, interpreted as colloid osmotic lysis. The relationships between the binding of the agent and its effects are as follows: a small, rapid initial uptake does not affect cation permeability; the subsequent slower uptake is associated with increased leakage of K⁺ and Na⁺; and the recovery at 25°C is associated with desorption of about half of the PCMBS due to competition by soluble thiol substances released into the medium from the cells. Desorption and “recovery” can be mimicked at any time by addition of small amounts of protein in the medium. The half of the PCMBS that cannot be desorbed is assumed to be bound by the hemoglobin inside the cell. The sulfhydryl groups involved in control of cation permeability constitute only a fraction of the total within the membrane (4–18%). They are located within the interior of the membrane separated from the medium and from the interior of the cell by diffusion barriers to PCMBS.     

Protein crowding in the inner mitochondrial membrane

The inner membrane of mitochondria is known for its low lipid-to-protein ratio. Calculations based on the size and the concentration of the principal membrane components, suggest about half of the hydrophobic volume of the membrane is occupied by proteins. Such high degree of crowding is expected to strain the hydrophobic coupling between proteins and lipids unless stabilizing mechanisms are in place. Both protein supercomplexes and cardiolipin are likely to be critical for the integrity of the inner mitochondrial membrane because they reduce the energy penalty of crowding.     

Effect of inductors of alkali cation permeability on cytochrome c bound to mitochondrial membrane]

The effect of inductors of alkali cation permeability--valinomycin, gramicidin A, gramicidin S and its N,N'-diacetyl derivative--on rat liver mitochondria during respiration has been studied. It is shown that valinomycin, gramycidin A and diacetylgramicidin S at optimal concentration for uncoupling cause two-phase activation of mitochondrial respiration and that this effect results from cytochrome c solubilization. Gramicidin S at optimal concentration cannot remove cytochrome c from the respirating mitochondria. It is suggested that this property of gramicidin S is owned to cytochrome c immobilisation in membrane, due to the effect of this compound.     

Electrophysiology of the inner mitochondrial membrane

The application of electrophysiological techniques to mitochondrial membranes has allowed the observation and partial characterization of several ion channels, including an ATP-sensitive K⁺-selective one, a high-conductance megachannel, a 107 pS anionic channel and three others studied at alkaline pH's. A reliable correlation with the results of non-electrophysiological studies has been obtained so far only for the first two cases. Activities presumed to be associated with the Ca²⁺ uniporter and with the adenine nucleotide translocator, as well as the presence of various other conductances have also been reported. The review summarizes the main properties of these pores and their possible relationship to permeation pathways identified in biochemical studies.     

Pathways shaping the mitochondrial inner membrane

Mitochondria are complex organelles with two membranes. Their architecture is determined by characteristic folds of the inner membrane, termed cristae. Recent studies in yeast and other organisms led to the identification of four major pathways that cooperate to shape cristae membranes. These include dimer formation of the mitochondrial ATP synthase, assembly of the mitochondrial contact site and cristae organizing system (MICOS), inner membrane remodelling by a dynamin-related GTPase (Mgm1/OPA1), and modulation of the mitochondrial lipid composition. In this review, we describe the function of the evolutionarily conserved machineries involved in mitochondrial cristae biogenesis with a focus on yeast and present current models to explain how their coordinated activities establish mitochondrial membrane architecture.     

Bilirubin increases mitochondrial inner membrane conductance

Bilirubin accumulates within, and induces loose coupling in, rat liver mitochondria. This state, characterized by a normal protonmotive force, but increased oxygen consumption and inner membrane conductance, could impair cellular energy metabolism. Loose coupling is observed at bilirubin concentrations (12-24 microM) attained in tissues of kernicteric animals.     

Mechanism accounting for the induction of nonspecific permeability of the inner mitochondrial membrane by hydroperoxides

The effect of antioxidants on the nonspecific permeability of the inner mitochondrial membrane induced by cumene hydroperoxide or Ca(2+) has been studied. Butylated hydroxytoluene, butylated hydroxyanisole and 2,2,5,7,8-pentamethyl-6-chromanol, taken at a concentration up to 50 microM, suppress the cumene hydroperoxide-induced accumulation of lipid peroxidation products. In the same range of concentrations, these antioxidants inhibit the activation of nonspecific permeability by cumene hydroperoxide or Ca(2+). Propyl gallate, being less effective under such conditions, fails to affect the induction of nonspecific permeability. Additionally, 2,2,5,7,8-pentamethyl-6-chromanol at a concentration decreasing the accumulation of lipid peroxidation products by 70% has been shown not to increase the lag period of nonspecific permeability induction. Higher antioxidant concentrations, while leading to an increase in the lag period of nonspecific permeability induction, cause but minor suppression of lipid peroxidation. From the results obtained we can assume that free radicals formed in the course of hydroperoxide decomposition or on mitochondrial redox complex interact directly with a system responsible for nonspecific permeability or with regulating components of this system.     

Modeling tubular shapes in the inner mitochondrial membrane

The inner mitochondrial membrane has been shown to have a novel structure that contains tubular components whose radii are of the order of 10 nm as well as comparatively flat regions. The structural organization of mitochondria is important for understanding their functionality. We present a model that can account, thermodynamically, for the observed size of the tubules. The model contains two lipid constituents with different shapes. They are allowed to distribute in such a way that the composition differs on the two sides of the tubular membrane. Our calculations make two predictions: (1) there is a pressure difference of 0.2 atmospheres across the inner membrane as a necessary consequence of the experimentally observed tubule radius of 10 nm, and (2) migration of differently shaped lipids causes concentration variations of the order of 7% between the two sides of the tubular membrane.     

Identification of anion and cation pathways in the inner mitochondrial membrane by patch clamping of mouse liver mitoplasts

Summary Alkalinization of the matrix side of the mitochondrial inner membrane by pH shifts from 6.8 to 8.3 caused a reversible increase in current of 3.2±0.2 pA (mean±se,n=21) at±40 mV measured using patch-clamp techniques. The current increase was reversed in a graded fashion by the addition of Mg²⁺ in 0.15m KCl corresponds to approximately 15 pS. Reversal potentials derived from whole patch currents indicated that the inner mitochondrial membrane was primarily cation selective at pH 6.8 with aP _k/P _Cl=32 (n=6). Treatment with alkaline pH (8.3) increased the current and anion permeability (P _K/P _Cl=16,n=6). The membrane becomes completely cation selective when low concentrations (12 m) of the drug propranolol are added. The amphiphilic drugs amiodarone (4 m), propranolol (70 m) and quinine (0.6mm) blocked almost all of the current. The pH-dependent current was also inhibited by tributyltin. These results are consistent with the presence of two pathways in the inner mitochondrial membrane. One is cation selective and generally open and the other is anion selective and induced by alkaline pH. The alkaline pH-activated channel likely corresponds to the inner membrane anion channel postulated by others from suspension studies.     

Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.