Electrical hypothesis of toxicity of the Cry toxins for mosquito larvae

Many electrical properties of insect larval guts have been studied, but their importance for toxicity of the Cry-type toxins has never been reported in the literature. In the present work, we observed potential-dependent permeabilization of plasma membrane by several polycationic peptides derived from the Cry11Bb protoxin. The peptide BTM-P1d, all D-type amino acid analogue of the earlier reported peptide BTM-P1, demonstrated high membrane-permeabilizing activity in experiments with isolated rat liver mitochondria, RBC (red blood cells) and mitochondria in homogenates of Aedes aegypti larval guts. Two larger peptides, BTM-P2 and BTM-P3, as well as the Cry11Bb protoxin treated with the protease extract of mosquito larval guts showed similar effects. Only protease-resistant BTM-P1d, in comparison with other peptides, displayed A. aegypti larval toxicity. Taking into account the potential-dependent mechanism of membrane permeabilization by studied fragments of the Cry11Bb protoxin and the literature data related to the distribution of membrane and transepithelial potentials in the A. aegypti larval midgut, we suggest an electrical hypothesis of toxicity of the Cry toxins for mosquito larvae. According to this hypothesis, the electrical field distribution is one of the factors determining the midgut region most susceptible for insertion of activated toxins into the plasma membrane to form pores. In addition, potential-dependent penetration of short active toxin fragments into the epithelial cells could induce permeabilization of mitochondria and subsequent apoptosis or necrosis.     

Ca2+ Induces a Cyclosporin A-Insensitive Permeability Transition Pore in Isolated Potato Tuber Mitochondria Mediated by Reactive Oxygen Species

Oxidative damage of mammalian mitochondria induced by Ca²⁺ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca²⁺ uniport, undergo inner membrane permeabilization when treated with Ca²⁺ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg²⁺ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca²⁺ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.     

Potential-dependent permeabilization of plasma membrane by the peptide BTM-P1 derived from the Cry11Bb1 protoxin

The peptide BTM-P1, which is derived from the amino acid sequence of the Cry11Bb1 protoxin, is able to permeabilize mitochondrial membranes and reveals antimicrobial activity. In this work we demonstrated that the permeabilizing activity of BTM-P1 for the plasma membrane of rat red blood cells increased in a dose-dependent manner for the concentration range of 1-4 μg/ml. Using osmotic protectants, the radius of pores formed at 4 μg/ml BTM-P1 was determined as 0.8 nm for 5 min hemolysis data, 0.7 nm for 5 min decrease in light dispersion of the cell suspension and 0.5 nm for the light dispersion slope measurements. The permeabilizing activity of 1 μg/ml peptide was increased by valinomycin-induced plasma membrane potential, especially under moderately hypotonic conditions. These results might explain the antimicrobial activity of BTM-P1 and support the hypothesis of potential-dependent and pro-apoptotic character of toxicity of naturally proteolysed Cry11Bb1 protoxin for epithelial cells of mosquito larvae midgut.     

Permeabilization of mitochondria and red blood cells by polycationic peptides BTM-P1 and retro-BTM-P1

Mitochondrial and plasma membrane permeabilization by polycationic peptides BTM-P1 and retro-BTM-P1 were studied. BTM-P1 was more active than its retro-analog. In the sucrose medium, the capacity of BTM-P1 to permeabilize mitochondria was lower than in salt media. In contrast, retro-BTM-P1 showed the lowest activity in the KCl medium. The efficacy of both peptides to permeabilize red blood cells was higher in the sucrose medium and depended on the nature of salt in high ionic strength media. BTM-P1, but not retro-BTM-P1, induced biphasic change in light dispersion of red blood cells with artificially generated high transmembrane potential: the initial phase of fast cell shrinkage preceded the subsequent phase of cell swelling. The shrunken red blood cells demonstrated increased sensitivity to BTM-P1 that might be explained by the cell suicide mechanism via phosphatidylserine exposure at the cell surface. As a working hypothesis, we assume that some peptide topology characteristics, such as the orientation and values of the total and local electrical dipole moments, interacting with the membrane dipole potential, as well as the asymmetric distribution of polar and non-polar side chains are important factors affecting the membrane-permeabilizing activity of polycationic peptides.     

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+.     

Rapid release of Mg(2+) from liver mitochondria by nonesterified long-chain fatty acids in alkaline media

Long-chain fatty acids induce a rapid release of Mg(2+) from both energized and nonenergized rat liver mitochondria suspended at pH 8 in isotonic saline but not sucrose media. The effect is observed only with fatty acids that possess protonophoric activity. The most active saturated fatty acids are myristic and palmitic, while the most active unsaturated acids are oleic, linolenic, and arachidonic. The rate of Mg(2+) release drastically decreases with decreasing medium pH to 7.2-7.6. However, at those pH values this rate is doubled by energization of mitochondria with respiratory substrates. Mg(2+) release is accompanied by cyclosporin A-insensitive large-amplitude swelling of mitochondria. This swelling is similar to that produced by the divalent metal ionophore A23187 and is interpreted as being due to activation of the inner membrane anion channel, the K(+) uniporter, and the K(+)/H(+) exchanger. In energized mitochondria, both swelling and Mg(2+) release are blocked by the exogenous K(+)/H(+) exchanger nigericin. It is proposed that fatty acids under conditions of alkaline mitochondrial matrix activate latent Mg(2+)-sensitive ion-conducting pathways in the inner mitochondrial membrane, which mediate swelling and Mg(2+) release. It is hypothesized that fatty acids activate an intrinsic Mg(2+)/H(+) exchanger that is related to, or identical with, the K(+)/H(+) exchanger.     

Synergistic inhibition of mitochondrial respiration by anticancer agent erucylphosphohomocholine and cyclosporin A

Alkylphosphocholines are a new class of anticancer agents. The mechanisms by which these drugs display their antitumor activities are not known. In this work, we show that erucylphosphohomocholine, a new antineoplastic compound, significantly decreased ATP synthesis in isolated rat liver mitochondria at a concentration of 50 microm or higher via permeabilization of the inner membrane. At a concentration of 25 microm, it induced a moderate swelling of mitochondria, a slight decrease of the inner membrane potential, and an increase in state 4 respiration without an essential influence on state 3 respiration or the outer membrane permeability to cytochrome c. We found that cyclosporin A did not prevent mitochondrial swelling induced by 25-100 microm erucylphosphohomocholine. Moreover, cyclosporin A induced a fast drop of the inner membrane potential in the presence of 25-50 microm erucylphosphohomocholine that seems to be due to a strong synergistic inhibition of the respiratory activity. The ratio of uncoupled to state 3 respiration rates increased from 1.3 +/- 0.1 with 25 microm erucylphosphohomocholine and from 1.5 +/- 0.1 with 1 microm cyclosporin A to 4.5 +/- 0.3 in the presence of both drugs. On the other hand, oligomycin or cyclosporin A protected certain cancer cell lines against erucylphosphohomocholine-induced apoptosis. This protection might be related to a prevention of cellular ATP hydrolysis by permeabilized mitochondria and to the inhibition of the classical permeability transition pore, respectively. Our findings provide new insight into the mechanisms by which these unusual alterations of mitochondria might be involved in anticancer activity of alkylphosphocholines.     

Biphasic oxidation of mitochondrial NAD(P)H

The redox state of mitochondrial pyridine nucleotides is known to be important for structural integrity of mitochondria. In this work, we observed a biphasic oxidation of endogenous NAD(P)H in rat liver mitochondria induced by tert-butylhydroperoxide. Nearly 85% of mitochondrial NAD(P)H was rapidly oxidized during the first phase. The second phase of NAD(P)H oxidation was retarded for several minutes, appearing after the inner membrane potential collapse and mitochondria swelling. It was characterized by disturbance of ATP synthesis and dramatic permeabilization of the inner membrane to pyridine nucleotides. The second phase was completely prevented by 0.5 microM cyclosporin A or 0.2 mM EGTA or was significantly delayed by 25 microM butylhydroxytoluene or trifluoperazine. The obtained data suggest that the second phase resulted from oxidation of the remaining NADH via the outer membrane electron transport system of permeabilized mitochondria, leading to further oxidation of the remaining NADPH in a transhydrogenase reaction.     

Possible Mechanism for Formation and Regulation of the Palmitate-Induced Cyclosporin A-Insensitive Mitochondrial Pore

The mechanism of the palmitate-induced opening of the mitochondrial Ca2+-dependent cyclosporin A (CsA)-insensitive pore was studied, as well as the influence on this process of well-known modulators of the CsA-sensitive Ca2+-dependent pore. Palmitic acid, which can bind Ca2+ with high affinity, induced the cyclosporin A-insensitive swelling of mitochondria, whereas palmitoleic and 2-bromopalmitic acids, which have no such affinity for Ca2+, failed to induce the pore opening. The palmitate-induced Ca2+-dependent swelling of mitochondria was not affected by a well-known inhibitor of the CsA-sensitive pore (ADP) and an activator of this pore (inorganic phosphate, P(i)). However, this swelling was inhibited by physiological concentrations of ATP ([I]50 = 1.3 mM), but 100 microM ATP increased by 30% the rate of mitochondria swelling if Ca2+ had been added earlier. The effects of ATP (inhibition and activation) manifested themselves from different sides of the inner mitochondrial membrane. Mg2+ inhibited the palmitate-induced Ca2+-dependent swelling of mitochondria with [I]50 = 0.8 mM. It is concluded that palmitic acid induces the opening of the CsA-insensitive pore due to its ability for complexing with Ca2+. A possible mechanism of the pore formation and the influence of some modulators on this process are discussed.     

Cd2+ -promoted mitochondrial permeability transition: a comparison with other heavy metals

We compared action of Cd(2+), Hg(2+), and Cu(2+) on isolated rat liver mitochondria in the absence of added Ca(2+) and P(i). The heavy-metal ions produced dose-dependently: (1) enhanced membrane permeabilization manifested in mitochondrial swelling and activation of basal respiration, (2) inhibition of uncoupler-stimulated respiration, and (3) membrane potential dissipation. Among the metals, Cu(2+) exhibited maximal stimulatory effect on basal respiration and minimal inhibitory action on DNP-uncoupled respiration whilst Cd(2+) promoted the strongest depression of uncoupled respiration and the largest swelling in NH(4)NO(3) medium. Dithiothreitol induced a basal respiration release if added after high [Cd(2+)] and [Hg(2+)], and the stimulation was CsA-insensitive.     

Effect of palmitoyl-CoA binding with adenine nucleotide translocase on energization of mitochondria]

Under conditions of inhibiting oxidative phosphorylation of oligomycin palmitoyl-CoA (p-CoA) decreases the rate of energy dependent reduction of acetoacetate and Ca2+-capacity of mitochondria in a phosphate medium. Energy independent osmotic swelling of mitochondria in NH4NO3, which depends on H+ permeability of the inner mitochondrial membrane is inhibited by ADP and acclereated by p-CoA. Carnitin and competitive ADP abolish all the effects of p-CoA. It is concluded that decreased energization induced by p-CoA is related to an increase in the inner mitochondrial membrane permeability b- H+ as a result of the inhibitor bindings with adenine nucleotide translocase.     

Avicins, natural anticancer saponins, permeabilize mitochondrial membranes

Avicins are a class of natural saponins with selective pro-apoptotic activity in cancer cells. In this work, we studied the influence of avicins on metabolic state of rat liver mitochondria. Avicin-treated mitochondria underwent a significant decrease in oxygen consumption rate that was completely restored by addition of exogenous cytochrome c. On the other hand, avicins increased the rotenone-insensitive oxidation of external NADH in the presence of exogenous cytochrome c, long before high amplitude swelling of mitochondria was observed. The increase in external NADH oxidation was cyclosporin A-insensitive. Avicin G significantly accelerated hydroperoxide-induced oxidation of mitochondrial endogenous NAD(P)H, the drop of the inner membrane potential and the high amplitude swelling of mitochondria. The obtained data might explain selective induction of apoptosis in tumor cells by avicins. Based on other studies showing that tumor cells generate hydroperoxides with a very high rate, avicins could provide a new strategy of anticancer therapy by sensitizing cells with high levels of reactive oxygen species to apoptosis.     

Cyclosporin A-sensitive permeability transition pore is involved in Cd(2+)-induced dysfunction of isolated rat liver mitochondria: doubts no more

There is dose-dependent Cd(2+)-evoked swelling of isolated rat liver mitochondria energized by complex I, II, or IV respiratory substrates in sucrose medium in the absence of added Ca(2+) and P(i), which is prevented by Sr(2+). Permeability transition effectors (ADP, CsA, EGTA, RR, DTT, ATR, P(i), and Ca(2+)) affect in a corresponding way Cd(2+)-promoted membrane permeabilization in NH(4)NO(3), KCl, and sucrose media. Maximal depression of Cd(2+)-induced swelling is achieved by simultaneous addition of ADP, Mg(2+), and CsA that produces either synergistic (NH(4)NO(3)) or additive (KCl and sucrose media) action. Sustained activation by low [Cd(2+)] of mitochondrial basal respiration in KCl medium is observed both in the absence and in the presence of rotenone and/or oligomycin but only in the latter case (rotenone+oligomycin) CsA inhibits completely Cd(2+) activation of St 4 respiration and partially reverses DNP-uncoupled respiration depressed by cadmium. Cd(2+) effects are discussed in terms of comparison with those of Zn(2+) and PhAsO.     

The effect of taurine on the ion transport system in mitochondria

The effect of taurine on the ATP-dependent mitochondrial swelling that characterizes the activity of mitochondrial ATP-dependent K⁺ channel and the formation of Ca²⁺-dependent pores, different in sensitivity to cyclosporin A, has been studied in rat liver mitochondria. It has been shown that taurine in micromolar concentrations (0.5–125 μM) stimulates the energy-dependent swelling of mitochondria. Taurine in physiological concentrations (0.5–20 mM) has no effect on the ATP-dependent swelling and the formation of cyclosporin A-insensitive Pal/Ca²⁺-activated pore in mitochondria. Taurine in these concentrations increased the rate of cyclosporin A-sensitive swelling of mitochondria induced by Ca²⁺ and P_i and reduced the Ca²⁺ capacity of mitochondria. The different effects of physiological taurine concentrations on the ATP-dependent transport of K⁺ and Ca²⁺ ions in mitochondrial membranes as compared with cell membranes are discussed.     

Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization

Photodynamic therapy (PDT), a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in human epidermoid carcinoma A431 cells. However, the precise mechanism of PDT-induced apoptosis is not well characterized. To dissect the pathways of PDT-induced apoptosis, we investigated the involvement of mitochondrial damage by examining a second generation photosensitizer, the silicon phthalocyanine 4 (Pc 4). By using laser-scanning confocal microscopy, we found that Pc 4 localized to cytosolic membranes primarily, but not exclusively, in mitochondria. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes when cells were exposed to Pc 4 and 670-675 nm light. This was followed by mitochondrial inner membrane permeabilization, depolarization and swelling, cytochrome c release, and apoptotic death. Desferrioxamine prevented mitochondrial ROS production and the events thereafter. Cyclosporin A plus trifluoperazine, blockers of the mitochondrial permeability transition, inhibited mitochondrial inner membrane permeabilization and depolarization without affecting mitochondrial ROS generation. These data indicate that the mitochondrial ROS are critical in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death during PDT with Pc 4.     

Influence of cholesterol on the formation of palmitate/Ca<Superscript>2+</Superscript>-activated pores in mitochondria and liposomes

The influence of cholesterol on the formation of a mitochondrial cyclosporin A-insensitive palmitate/Ca²⁺-activated pore has been studied. Loading of mitochondrial membranes with cholesterol increases the rate of mitochondrial swelling induced by palmitic acid (≥20 μM) and Ca²⁺ (30 μM). This effect is not related to changes in the functional activity of organelles, since cholesterol does not influence the mitochondrial respiration in different metabolic states. At the same time, palmitate/Ca²⁺-induced permeabilization of azolectin/cholesterol liposomes is more pronounced than that of azolectin liposomes. In the liposomal membrane, Ca²⁺ induces phase separation of palmitic acid into distinct membrane domains; the presence of cholesterol in membranes enhances this effect.     

The role of mitochondrial palmitate/Ca<Superscript>2+</Superscript>-activated pore in palmitate-induced apoptosis

The evidence of possible involvement of the mitochondrial cyclosporin A-insensitive palmitate/Ca²⁺-activated pore in palmitate-induced apoptosis is presented. It has been established that the opening of the palmitate/Ca²⁺-activated pore results in the high-amplitude swelling of mitochondria and the release of the apoptosis-inducing factor from organelles. These processes are accompanied by a short-term slight decrease of membrane potential, which recovers in 1 min. The possible role of the palmitate/Ca²⁺-activated pore in the induction of palmitate-induced apoptosis is discussed.     

Osmotically-induced alterations in volume and ultrastructure of mitochondria isolated from rat liver and bovine heart

Detailed studies correlating changes in mitochondrial optical density, packed volume, and ultrastructure associated with osmotically-induced swelling were performed. Various swelling states were established by incubating mitochondria (isolated in 0.25 M sucrose) at 0°C for 5 min in series of KCl and sucrose solutions ranging in tonicity from 250 to 3 milliosmols. Reversibility of swelling was determined by examining mitochondria exposed to 250 milliosmols media after they had been induced to swell. Swelling induced by lowering the ambient tonicity to approximately 130 (liver mitochondria) and 90 (heart mitochondria) milliosmols involves primarily swelling of the inner compartment within the intact outer membrane. Decreasing the ambient tonicity beyond this level results in rupture of the outer membrane and expansion of the inner compartment through the break. The maximum extent of swelling, corresponding with complete unfolding of the cristae and an increase in over-all mitochondrial volume of approximately 6-fold (liver mitochondria) and 11-fold (heart mitochondria), is reached at approximately 15 (liver mitochondria) and 3 (heart mitochondria) milliosmols. Exposure of liver mitochondria to media of lower tonicity results in irreversibility of inner compartment swelling and escape of matrix material. These changes appear to result from increased inner membrane permeability, possibly due to stretching.     

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.     

BMAP-28, an antibiotic peptide of innate immunity, induces cell death through opening of the mitochondrial permeability transition pore

BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca(2+) and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability.