Dehydroepiandrosterone as an inducer of mitochondrial permeability transition

This paper reports an investigation upon the effect of dehydroepiandrosterone (DHEA) on some mitochondrial membrane functions, such as electron transport, transmembrane electric gradient and calcium permeability. It was found that the hormone induced the efflux of accumulated matrix Ca²⁺, inhibited Site I of the respiratory chain, as well as bringing about the collapse of the transmembrane potential, and mitochondrial swelling. Taking into account that cyclosporin A (CSA) inhibited Ca²⁺ release and the collapse of the transmembrane potential, it is concluded that the hormone may induce the opening of a non-specific transmembrane pore. The mechanism of pore opening is ascribed to peroxidation of the membrane lipid bilayer. It should be mentioned that estrone, even at the concentration of 200 μM, failed to reproduce the behavior of dehydroepiandrosterone on mitochondrial functions.     

Flufenamic acid as an inducer of mitochondrial permeability transition

To assess the mechanism by which mitochondrial permeability transition (MPT) is induced by the nonpolar carboxylic acids, we investigated the effects of flufenamic acid (3-trifluoromethyl diphenylamine-2-carboxylic acid, FA) on mitochondrial respiration, electrical transmembrane potential difference (), osmotic swelling, Ca²⁺ efflux, NAD(P)H oxidation and reactive oxygen species (ROS) generation. Succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 10 M Ca²⁺, 5 M ruthenium red (RR) or 1 M cyclosporin A (CsA) were used. The dose response-curves for both respiration release and dissipation were nearly linear, presenting an IC₅₀ of approximately 10 M and reaching saturation within 25-50 M, indicating that FA causes mitochondrial uncoupling by a protonophoric mechanism. Within this same concentration range FA showed the ability to induce MPT in energized mitochondria incubated with 10 M Ca²⁺, followed by dissipation and Ca²⁺ efflux, and even in deenergized mitochondria incubated with 0.5 mM Ca²⁺. ADP, Mg²⁺, trifluoperazine (TFP) and N-ethylmaleimide (NEM) reduced the extent of FA-promoted swelling in energized mitochondria by approximately one half, whereas dithiothreitol (DTT) slightly enhanced it. NAD(P)H oxidation and ROS generation (H₂O₂ production) by mitochondria were markedly stimulated by FA; these responses were partly prevented by CsA, suggesting that they may be implicated as both a cause and effect of FA-induced MPT. FA incubated with mitochondria under swelling assay conditions caused a decrease of approximately 40% in the content of protein thiol groups reacting with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB). The present results are consistent with a ROS-intermediated sensitization of MPT by a direct or indirect FA interaction with inner mitochondrial membrane at a site which is in equilibrium with the NAD(P)H pool, namely thiol groups of integral membrane proteins.     

Ion permeability of bilayer membranes formed from synthetic phospholipids in the phase transition region

Ion permeability of black lipid membranes formed from synthetic phospholipids has been studied. The resistance of BLM formed from phosphatidylcholine, tiophosphatidylcholine, threealkylphosphate and threealkyltiophosphate was 10(7)--10(8) Ohm.cm2. It was shown that the membrane potential of the 10--30 mV arised in KCl gradient indicating the preference cation conductance in synthetic lipid membranes. A sharp decrease of the membrane conductance near to the phase transition temperature was discovered. The change of conductance by phase transition temperature was sensitive to chemical nature of the polar head of phospholipids used.     

Zinc as an inducer of the membrane permeability transition in rat liver mitochondria

It is shown that 2-10 microM 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 by tert-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 microM. 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.     

Electrophysiological properties of the mitochondrial permeability transition pores: Channel diversity and disease implication

The mitochondrial permeability transition pore (mPTP) is a channel that, when open, is responsible for a dramatic increase in the permeability of the mitochondrial inner membrane, a process known as the mitochondrial permeability transition (mPT). mPTP activation during Ca²⁺ dyshomeostasis and oxidative stress disrupts normal mitochondrial function and induces cell death. mPTP opening has been implicated as a critical event in many diseases, including hypoxic injuries, neurodegeneration, and diabetes. Discoveries of recent years indicate that mPTP demonstrates very complicated behavior and regulation, and depending on specific induction or stress conditions, it can function as a high-conductance pore, a small channel, or a non-specific membrane leak. The focus of this review is to summarize the literature on the electrophysiological properties of the mPTP and to evaluate the evidence that it has multiple molecular identities. This review also provides perspective on how an electrophysiological approach can be used to quantitatively investigate the biophysical properties of the mPTP under physiological, pharmacological, pathophysiological, and disease conditions.     

Uterine vascular permeability, blood flow and extracellular fluid space during implantation in rats

Vascular permeability to plasma proteins in uterine implantation and non-implantation sites (i.e. dye sites and non-dye sites) was assessed quantitatively by a method which accounts for steady-state volumes of distribution. Extracellular fluid volume and uterine blood flow were also determined. On both the evening of Day 5 and the morning of Day 6, vascular permeability to 125I-labelled human serum albumin, extracellular fluid volume and blood flow were significantly increased in implantation sites compared to non-implantation sites. Vascular permeability in implantation sites was increased significantly between Days 5 and 6, whereas that in non-implantation sites was unchanged. This increase in vascular permeability between Days 5 and 6 was not accompanied by further increases in extracellular fluid volume and blood flow. This result shows a dissociation between vascular permeability and extracellular fluid volume immediately after the onset of implantation and raises important questions as to whether the rat uterus undergoes a truly oedematous response at implantation as has been generally accepted.     

The permeability of reconstituted nuclear pores provides direct evidence for the selective phase model

Nuclear pore complexes (NPCs) maintain a permeability barrier between the nucleus and the cytoplasm through FG-repeat-containing nucleoporins (Nups). We previously proposed a "selective phase model" in which the FG repeats interact with one another to form a sieve-like barrier that can be locally?disrupted by the binding of nuclear transport receptors (NTRs), but not by inert macromolecules, allowing selective passage of NTRs and associated cargo. Here, we provide direct evidence for this model in a physiological context. By using NPCs reconstituted from Xenopus laevis egg extracts, we show that Nup98 is essential for maintaining the permeability barrier. Specifically, the multivalent cohesion between FG repeats is required, including cohesive FG repeats close to the anchorage point to the NPC scaffold. Our data exclude alternative models that are based solely on an interaction between the FG repeats and NTRs and indicate that the barrier is formed by a sieve-like FG hydrogel.     

Low pH as an inhibiting factor in the transition from mesophilic to thermophilic phase in composting

During composting of household waste, the acidity of the material affects the process during the initial phase of rising temperature. In this study, the effects of temperature (36-46 degrees C) and pH (4.6-9.2) on the respiration rate during the early phase of composting were investigated in two different composts. A respiration method where small compost samples were incubated at constant temperature was used. The respiration rate was strongly reduced at 46 degrees C and pH below 6, compared to composts with a higher pH or lower temperature. The combination of high temperature and low pH is a possible adverse factor in large-scale composting of food waste.     

Real-time flow cytometry analysis of permeability transition in isolated mitochondria

Mitochondrial membrane permeabilization (MMP) is a key event in necrotic and (intrinsic) apoptotic processes. MMP is controlled by a few major rate-limiting events, one of which is opening of the permeability transition pore (PTP). Here we develop a flow cytometry (FC)-based approach to screen and study inducers and blockers of MMP in isolated mitochondria. Fixed-time and real-time FC permits to co-evaluate and order modifications of mitochondrial size, structure and inner membrane (IM) electrochemical potential (DeltaPsi(m)) during MMP. Calcium, a major PTP opener, and alamethicin, a PTP-independent MMP inducer, trigger significant mitochondrial forward scatter (FSC) increase and side scatter (SSC) decrease, correlating with spectrophotometrically detected swelling. FC-based fluorescence detection of the DeltaPsi(m)-sensitive cationic lipophilic dye JC-1 permits to detect DeltaPsi(m) variations induced by PTP openers or specific inducers of inner MMP such as carbonylcyanide m-chlorophenylhydrazone (mClCCP). These simple, highly sensitive and quantitative FC-based methods will be pertinent to evaluate compounds for their ability to control MMP.     

Bistability in apoptosis: roles of bax, bcl-2, and mitochondrial permeability transition pores

We propose a mathematical model for mitochondria-dependent apoptosis, in which kinetic cooperativity in formation of the apoptosome is a key element ensuring bistability. We examine the role of Bax and Bcl-2 synthesis and degradation rates, as well as the number of mitochondrial permeability transition pores (MPTPs), on the cell response to apoptotic stimuli. Our analysis suggests that cooperative apoptosome formation is a mechanism for inducing bistability, much more robust than that induced by other mechanisms, such as inhibition of caspase-3 by the inhibitor of apoptosis (IAP). Simulations predict a pathological state in which cells will exhibit a monostable cell survival if Bax degradation rate is above a threshold value, or if Bax expression rate is below a threshold value. Otherwise, cell death or survival occur depending on initial caspase-3 levels. We show that high expression rates of Bcl-2 can counteract the effects of Bax. Our simulations also demonstrate a monostable (pathological) apoptotic response if the number of MPTPs exceeds a threshold value. This study supports our contention, based on mathematical modeling, that cooperativity in apoptosome formation is critically important for determining the healthy responses to apoptotic stimuli, and helps define the roles of Bax, Bcl-2, and MPTP vis-à-vis apoptosome formation.     

A theoretical study of the distribution of substances around roots resulting from simultaneous diffusion and mass flow

Summary The change in concentration of a solute in soil, moving near the surface of a root by both mass flow and diffusion, has been calculated by a numerical method with a computer. The effect of change in the plant controlled variables v₀ (the solvent flux at the root surface) and k (the root absorbing power), and the soil variables b (the buffer power) and D (the diffusion coefficient) are described in turn.The concentration at the root surface, relative to the undisturbed soil solution, approaches a limiting value v₀/k. As v₀ is increased, the limiting value is approached more rapidly, and the zone of disturbance is more compressed. A steady state is reached if r₀v₀/bD>2, but if r₀v₀/bD<2 the disturbance continues to spread outwards even though the concentration at the root surface has nearly attained its limiting value.As k is increased, other factors being constant, the limiting relative concentration at the root surface is approached more rapidly, but the spread of the disturbance away from the root is little affected.As Db is decreased, corresponding to a decrease in soil moisture, the concentration at the root surface reaches its limit more rapidly and the zone of disturbance is compressed.If, because of increase in the concentration at the root surface, the efficiency of root absorption declines, the relative concentration will exceed v₀/k, and may reach no limit — at least until the assumptions of the model used break down.     

Water permeability of isolated cuticular membranes: The effect of pH and cations on diffusion,hydrodynamic permeability and size of polar pores in the cutin matrix

Summary The upper astomatous cuticle of Citrus aurantium L. leaves was isolated enzymatically or chemically, extracted with lipid solvents and used for the determination of water diffusion (P _d ) and osmotic water permeability (P _f ). The water permeability was strongly dependent on the pH value and the cations of the buffer solutions. In presence of monovalent alkali metal ions P _d increased almost five fold between pH 3 and 11. The shape of the plot P _d vs. pH suggests the presence of 3 different dissociable groups fixed to the membrane matrix. They are tentatively identified as two carboxyl groups dissociating between pH 3 to 6 and 6 to 9, respectively, and as phenolic hydroxyl groups dissociating above pH 9. The carboxyl group dissociating between pH 6 and 9 discriminated between alkali metal ions according to their ionic radius. Water permeability was lowest in the Li⁺ from and increased in the order Li⁺+++. The water permeability of membranes in Ca²⁺ form was only slightly higher than that of membranes in H⁺ form and little dependent on pH. The energy of activation which amounted to 13 kcal mol^–1 was constant over the temperature range of 5 to 40°C and pH independent. Since P _f was greater than P _d it was concluded that the cutin matrix contained polar pores and that water transport caused by a chemical potential gradient was both by diffusion and by viscous flow. The porous nature of the membranes was also confirmed by the fact that they are permselective according to size of the permeating molecule. Using the empirical equations of Paganelli and Solomon (1957) and Nevis (1958) the equivalent radius of the pores was estimated to be 0.46 and 0.45 nm, respectively. This estimate is in good agreement with the observations that (a) [¹⁴C]urea (molecular radius r _s =0.264 nm) and [³H]glucose (r _s =0.444 nm) penetrated the membranes and (b) the reflection coefficient was equal to one for raffinose (r _s =0.654 nm) and sucrose (r _s =0.555 nm) but 0.95 for glucose and 0.78 for urea. Both, the reflection coefficient and the pore radius estimates were pH independent, hence the increase in water permeability with increasing pH was due to an increase in the number of pores per unit area (1 cm²) from 5x10¹⁰ at pH 3 to 15.8x10¹⁰ at pH 9.Abbreviations THO tritiated water - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES (N-morpholino) ethane sulphic acid - SADH succinic acid 2,2-dimethyl hydrazide     

On the large error introduced in the estimate of the density of membrane pores from permeability measurements when diffusion in "unstirred layer" within the cells is disregarded

After the development of the "black lipid membrane" techniques, studies of the permeability of labeled water and nonelectrolytes across these artificial membranes have yielded permeability constants comparable in magnitude to those obtained from tracer studies of living cell membranes. This general agreement has affirmed the belief that the living cell membranes are indeed closely similar to these bilayer phospholipid membranes. In this report, we draw attention to a hidden assumption behind such comparisons made: the assumption that labeled material passing through the cell membrane barriers instantly reaches diffusion equilibrium inside the cell. The permeability constants to labeled water (and nonelectrolytes) across lipid layers were obtained using setups in which the lipid membrane was sandwiched between aqueous compartments both of which were vigorously stirred. In studies of permeability of living cell membranes only the outside solution was stirred, the intracellular water remained stationary. Yet the calculations of permeability constants of the cell membrane were made with the tacit assumption, that once the labeled materials pass through the cell membrane, they were instantly mixed with the entire cell contents as if a stirrer operating at infinite speed had been present inside the cells. Ignoring this unstirred condition of the intracellular water, in fact, lumped all the real-life delay due to diffusion in the cytoplasm and added it to the resistance to diffusion of the membrane barrier. The result is an estimated membrane permeability to labeled water (and nonelectrolytes) many times slower than it actually is. The present report begins with a detailed analysis of a specific case: tritiated water diffusion from giant barnacle muscle fibers and two non-living models, one real, one imagined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turbidity changes of lipid vesicles near the phase transition temperature as an indication of fusion

Sonicated liposomes of dipalmitoyl phosphatidylcholine show sharp turbidity changes on heating at two distinct temperatures. A decrease in turbidity at the lower temperature (approx. 37°C) is thought to be associated with the phase transition of small vesicles and a decrease at about 44°C with larger vesicles or multilayer. An increase of turbidity between 38 and 43°C is attributed to the fusion of small vesicles. The turbidity changes were studied under various modes of vesicle preparation to confirm the interpretation of the turbidity data. Alternate interpretations are discussed.     

Function of Nicotiana tabacum aquaporins as chloroplast gas pores challenges the concept of membrane CO2 permeability

Photosynthesis is often limited by the rate of CO(2) diffusion from the atmosphere to the chloroplast. The primary resistances for CO(2) diffusion are thought to be at the stomata and at photosynthesizing cells via a combination resulting from resistances of aqueous solution as well as the plasma membrane and both outer and inner chloroplast membranes. In contrast with stomatal resistance, the resistance of biological membranes to gas transport is not widely recognized as a limiting factor for metabolic function. We show that the tobacco (Nicotiana tabacum) plasma membrane and inner chloroplast membranes contain the aquaporin Nt AQP1. RNA interference-mediated decreases in Nt AQP1 expression lowered the CO(2) permeability of the inner chloroplast membrane. In vivo data show that the reduced amount of Nt AQP1 caused a 20% change in CO(2) conductance within leaves. Our discovery of CO(2) aquaporin function in the chloroplast membrane opens new opportunities for mechanistic examination of leaf internal CO(2) conductance regulation.     

Change of shoot architecture during juvenile-to-adult phase transition in soybean

Juvenile-to-adult phase change is an indispensable event which guarantees a successful life cycle. Phase change has been studied in maize, Arabidopsis and rice, but is mostly unknown in other species. Soybean/Fabaceae plants undergo drastic changes of shoot architecture at the early vegetative stage including phyllotactic change and leaf type alteration from simple to compound. These characteristics make soybean/Fabaceae plants an interesting taxon for investigating vegetative phase change. Following the expansion of two cotyledons, two simple leaves simultaneously emerge in opposite phyllotaxy. The phyllotaxy of the third and fourth leaves is not fixed; both opposite and distichous phyllotaxis are observed within the same population. Leaves were compound from the third leaf. But the third leaf was rarely simple. Morphological and quantitative changes in early vegetative phase were recognized in leaf size, leaf shape, number of trichomes, stipule size and shape, and shoot meristem shape. Two microRNA genes, miR156 and miR172, are known to be associated with vegetative phase change. Examination of the expression level revealed that miR156 expression was high in the first two leaves and subsequently down-regulated, and that of miR172 showed the inverse expression pattern. These expression patterns coincided with the case of other species. Taken all data together, the first and second leaves represent juvenile phase, the fifth and upper leaves adult phase, and the third and fourth leaves intermediate stage. Further investigation of soybean phase change would give fruitful understandings on plant development.     

Mitochondrial permeability transition as a novel principle of hepatorenal toxicity in vivo

Atractyloside (Atr) binds to the adenine nucleotide translocator (ANT) and inhibits ANT-mediated ATP/ADP exchange on the inner mitochondrial membrane. In addition, Atr can trigger opening of a non-specific ion channel, within the ANT-containing permeability transition pore complex (PTPC), which is subject to redox regulation and inhibited by cyclosporin A (CsA). Here we show that the cytotoxic effects of Atr, both in vivo and in vitro, are determined by its capacity to induce PTPC opening and consequent mitochondrial membrane permeabilization (MMP). Thus, the Atr-induced MMP and death of cultured liver cells are both inhibited by CsA as well as by glutathione (GSH) and enhanced by GSH depletion. Similarly, the hepatorenal toxicity of Atr, assessed in vivo, was reduced by treating mice with CsA or a diet rich in sulfur amino acids, a regime which enhances mitochondrial GSH levels. Atr injection induced MMP in hepatocytes and proximal renal tubular cells, and MMP was reduced by either CsA or GSH. Acetaminophen (paracetamol)-induced acute poisoning was also attenuated by CsA and GSH, both in vitro and in vivo. Altogether these data indicate that PTPC-mediated MMP may determine the hepatorenal toxicity of xenobiotics in vivo.