ANT-Mediated Inhibition of the Permeability Transition Pore Alleviates Palmitate-Induced Mitochondrial Dysfunction and Lipotoxicity.

Hyperlipidemia is a major risk factor for vascular lesions in diabetes mellitus and other metabolic disorders, although its basis remains poorly understood. One of the key pathogenetic events in this condition is mitochondrial dysfunction associated with the opening of the mitochondrial permeability transition (MPT) pore, a drop in the membrane potential, and ROS overproduction. Here, we investigated the effects of bongkrekic acid and carboxyatractyloside, a potent blocker and activator of the MPT pore opening, respectively, acting through direct interaction with the adenine nucleotide translocator, on the progression of mitochondrial dysfunction in mouse primary lung endothelial cells exposed to elevated levels of palmitic acid. Palmitate treatment (0.75 mM palmitate/BSA for 6 days) resulted in an 80% decrease in the viability index of endothelial cells, which was accompanied by mitochondrial depolarization, ROS hyperproduction, and increased colocalization of mitochondria with lysosomes. Bongkrekic acid (25 µM) attenuated palmitate-induced lipotoxicity and all the signs of mitochondrial damage, including increased spontaneous formation of the MPT pore. In contrast, carboxyatractyloside (10 µM) stimulated cell death and failed to prevent the progression of mitochondrial dysfunction under hyperlipidemic stress conditions. Silencing of gene expression of the predominate isoform ANT2, similar to the action of carboxyatractyloside, led to increased ROS generation and cell death under conditions of palmitate-induced lipotoxicity in a stably transfected HEK293T cell line. Altogether, these results suggest that targeted manipulation of the permeability transition pore through inhibition of ANT may represent an alternative approach to alleviate mitochondrial dysfunction and cell death in cell culture models of fatty acid overload.

Effect of 2-aminoethoxydiphenyl borate on the functions of mouse skeletal muscle mitochondria.

This work examined the effect of 2-aminoethoxydiphenyl borate (2-APB) on the functioning of isolated mouse skeletal muscle mitochondria and modeled its putative interaction with mitochondrial proteins. We have shown that 2-APB is able to dose-dependently suppress mitochondrial respiration in state 3 and 3UDNP driven by substrates of complex I and II. This effect of 2-APB was accompanied by a slight dose-dependent decrease in mitochondrial membrane potential and appears to be due to inhibition of complex I and complex III of the electron transport chain (ETC) with IC50 values of 200 and 120 µM, respectively. The results of molecular docking identified putative 2-APB interaction sites in these ETC complexes. 2-APB was shown to dose-dependently inhibit both mitochondrial Ca2+ uptake and Ca2+ efflux, which seems to be caused by a decrease in the membrane potential of the organelles. We have found that 2-APB has no significant effect on mitochondrial calcium retention capacity. On the other hand, 2-APB exhibited antioxidant effect by reducing mitochondrial hydrogen peroxide production but without affecting superoxide generation. It is concluded that the effect of 2-APB on mitochondrial targets should be taken into account when interpreting the results of cell and in vivo experiments.

δ-Opioid Receptor as a Molecular Target for Increasing Cardiac Resistance to Reperfusion in Drug Development.

An analysis of published data and the results of our own studies reveal that the activation of a peripheral δ2-opioid receptor (δ2-OR) increases the cardiac tolerance to reperfusion. It has been found that this δ2-OR is localized in cardiomyocytes. Endogenous opioids are not involved in the regulation of cardiac resistance to reperfusion. The infarct-limiting effect of the δ2-OR agonist deltorphin II depends on the activation of several protein kinases, including PKCδ, ERK1/2, PI3K, and PKG. Hypothetical end-effectors of the cardioprotective effect of deltorphin II are the sarcolemmal KATP channels and the MPT pore.

The Role of NO Synthase, MPT pore, and Protein Kinase A in the Cardioprotective Effect of the Opioid Receptor Agonist Deltorphin II.

In male Wistar rats, coronary occlusion (45 min) and reperfusion (120 min) were modeled. Selective δ2-opioid receptor agonist (deltorphin II, 0.12 mg/kg) was administered intravenously 5 min before reperfusion; NO synthase inhibitor (L-NAME, 10 mg/kg), MPT pore blocker (atractyloside, 5 mg/kg), and protein kinase A inhibitor (H-89, 10 µg/kg) were administered intravenously 10 min before reperfusion. Deltorphin II administered before reperfusion led to a 2-fold decrease in the infarct size. The infarct-limiting effect of deltorphin II was associated with blockade of MPT pore. Protein kinase A and NO synthase were not involved in the cardioprotective effect of deltorphin II.

BKCa Activator NS1619 Improves the Structure and Function of Skeletal Muscle Mitochondria in Duchenne Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive hereditary disease caused by the absence of the dystrophin protein. This is secondarily accompanied by a dysregulation of ion homeostasis, in which mitochondria play an important role. In the present work, we show that mitochondrial dysfunction in the skeletal muscles of dystrophin-deficient mdx mice is accompanied by a reduction in K+ transport and a decrease in its content in the matrix. This is associated with a decrease in the expression of the mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) in the muscles of mdx mice, which play an important role in cytoprotection. We observed that the BKCa activator NS1619 caused a normalization of mitoBKCa expression and potassium homeostasis in the muscle mitochondria of these animals, which was accompanied by an increase in the calcium retention capacity, mitigation of oxidative stress, and improvement in mitochondrial ultrastructure. This effect of NS1619 contributed to the reduction of degeneration/regeneration cycles and fibrosis in the skeletal muscles of mdx mice as well as a normalization of sarcomere size, but had no effect on the leakage of muscle enzymes and muscle strength loss. In the case of wild-type mice, we noted the negative effect of NS1619 manifested in the inhibition of the functional activity of mitochondria and disruption of their structure, which, however, did not significantly affect the state of the skeletal muscles of the animals. This article discusses the role of mitoBKCa in the development of DMD and the prospects of the approach associated with the correction of its function in treatments of this secondary channelopathy.

An in vivo study of the toxic effects of triclosan on Xenopus laevis (Daudin, 1802) frog: Assessment of viability, tissue damage and mitochondrial dysfunction.

The present study describes the in vivo effect of triclosan on the frog Xenopus laevis (Daudin, 1802). We have found a dose-dependence of the effect of triclosan on the survival of frogs. At a dose of 2 mg/L, the death of frogs was observed already on the 4th day of the experiment, while at a concentration of 0.5 mg/L, the frogs remained viable for 11 days. Triclosan caused damage to the liver tissue, which was expressed in an increase in the area of hemorrhage and the number of melanomacrophage centers. 0.5 mg/L of this agent did not affect the number of frog red blood cells, but reduced their osmotic resistance. Keeping animals in water containing triclosan (0.5 mg/L for 96 h) led to the suppression of the state 3 respiration rate of frog liver mitochondria. This effect was accompanied by suppression of the combined activity of complexes II and III of the mitochondrial respiratory chain. In parallel with this, we observed a reduction in the Ca2+ retention capacity of frog liver mitochondria, indicating a decrease in the resistance of organelles to mitochondrial permeability transition pore opening. The paper discusses the effects of triclosan on aquatic organisms.

Conjugation of Aminoadamantane and γ-Carboline Pharmacophores Gives Rise to Unexpected Properties of Multifunctional Ligands.

A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer's disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure-activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced ß-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.

Diabetes Mellitus, Mitochondrial Dysfunction and Ca2+-Dependent Permeability Transition Pore.

Diabetes mellitus is one of the most common metabolic diseases in the developed world, and is associated either with the impaired secretion of insulin or with the resistance of cells to the actions of this hormone (type I and type II diabetes, respectively). In both cases, a common pathological change is an increase in blood glucose-hyperglycemia, which eventually can lead to serious damage to the organs and tissues of the organism. Mitochondria are one of the main targets of diabetes at the intracellular level. This review is dedicated to the analysis of recent data regarding the role of mitochondrial dysfunction in the development of diabetes mellitus. Specific areas of focus include the involvement of mitochondrial calcium transport systems and a pathophysiological phenomenon called the permeability transition pore in the pathogenesis of diabetes mellitus. The important contribution of these systems and their potential relevance as therapeutic targets in the pathology are discussed.

Itaconic acid impairs the mitochondrial function by the inhibition of complexes II and IV and induction of the permeability transition pore opening in rat liver mitochondria.

Itaconic acid (methylene-succinic acid, ItA) is an unsaturated dicarboxylic acid that is secreted by mammalian macrophages in response to a pro-inflammatory stimulus and shows an anti-inflammatory/antibacterial effect. Being a mitochondrial metabolite, it exhibits an inhibitory activity on succinate dehydrogenase and subsequently induces mitochondrial dysfunction. The present study has shown that ItA dose-dependently inhibited ADP- and DNP-stimulated (uncoupled) respiration of rat liver mitochondria energized with succinate. This effect of ItA could be related to the suppression of the activity of complex II and the combined activity of complexes II + III of the respiratory chain. At the same time, ItA had no effect on the activity of the dicarboxylate carrier, which catalyzes the transport of succinate across the inner mitochondrial membrane. It was found that 4 mM ItA diminished the rates of ADP- and DNP-stimulated mitochondrial respiration supported by the substrates of complex I glutamate and malate. A study of the effect of ItA on the activity of complexes of the respiratory chain showed that it decreases the activity of complex IV. It was observed that 4 mM ItA inhibited the rate of H2O2 production by mitochondria. At the same time, ItA promoted the opening of the cyclosporin A-sensitive Ca2+-dependent permeability transition pore. The latter was revealed as the decrease in the calcium retention capacity of mitochondria and the stimulation of release of cytochrome c from the organelles. ItA by itself promoted the cytochrome c release from mitochondria. Possible mechanisms of the effect of ItA on mitochondrial function are discussed.

Transport of Ca2+ and Ca2+-Dependent Permeability Transition in Rat Liver Mitochondria under the Streptozotocin-Induced Type I Diabetes.

Although diabetes mellitus is known to be a disease associated with mitochondrial dysfunction, not everything is clear about mitochondrial Ca2+ transport and Ca2+-induced permeability transition in diabetic cells. The objective of this work was to study the operation of MCU and Ca2+-dependent mitochondrial permeabilization in the liver cells of Sprague-Dawley rats under the streptozotocin-induced type I diabetes. It was shown that two weeks after the induction of diabetes, the rate of Ca2+ uptake by the mitochondria of diabetic animals increased ~1.4-fold. The expression of MCU and MICU1 subunits did not change, yet the quantity of dominant-negative MCUb channel subunits was almost twice as lower. The organelles also became more resistant to the induction of CsA-sensitive MPT pore and less resistant to the induction of CsA-insensitive palmitate/Ca2+-induced pore. The mitochondria of diabetic liver cells also showed changes in the lipid matrix of their membranes. The content of fatty acids in the membranes grew, and microviscosity of the lipid bilayer (assessed with laurdan) increased. At the same time, lipid peroxidation (assessed by the production of malonic dialdehyde) was stimulated. The paper discusses the consequences of the diabetes-related changes in mitochondria in the context of cell physiology.

Effect of bedaquiline on the functions of rat liver mitochondria.

The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50 µM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V3 and VDNP. At the same time, at concentrations below 50 µM, BDQ slightly stimulated respiration with substrates of complex I in the state V2. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II + III of the mitochondrial transport chain. It was discovered that at concentrations up to 10 µM, BDQ inhibited H2O2 production in mitochondria. BDQ (10-50 µM) suppressed the opening of Ca2+-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca2+/Pi-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca2+ capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K+ transport, which was evaluated by the energy-dependent swelling of mitochondria in a K+ buffer and DNP-induced K+ efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed.

Putative roles of mitochondrial Voltage-Dependent Anion Channel, Bcl-2 family proteins and c-Jun N-terminal Kinases in ischemic stroke associated apoptosis.

There is a constant need for better stroke treatments. Neurons at the periphery of an ischemic stroke affected brain tissue remains metabolically active for several hours or days after stroke onset. They later undergo mitochondrion-mediated apoptosis. It has been found that inhibiting apoptosis in the peripheral ischemic neurons could be very effective in the prevention of stroke progression. During stroke associated apoptosis, cytosolic c-Jun N-terminal Kinases (JNKs) and Bcl-2 family proteins translocate towards mitochondria and promote cytochrome c release by interacting with the outer mitochondrion membrane associated proteins. This review provides an overview of the plausible interactions of the outer mitochondrial membrane Voltage Dependent Anion Channel, Bcl-2 family proteins and JNKs in cytochrome c release in the peripheral ischemic stroke associated apoptotic neurons. The review ends with a note on designing new anti-stroke treatments.

Remote ischemic preconditioning of cardiomyocytes inhibits the mitochondrial permeability transition pore independently of reduced calcium-loading or sarcKATP channel activation.

Ischemic preconditioning (IPC) inhibits Ca(2+)-loading during ischemia which contributes to cardioprotection by inhibiting mechanical injury due to hypercontracture and biochemical injury through mitochondrial permeability transition (MPT) pores during reperfusion. However, whether remote-IPC reduced Ca(2+)-loading during ischemia and its subsequent involvement in inhibiting MPT pore formation during reperfusion has not been directly shown. We have developed a cellular model of remote IPC to look at the impact of remote conditioning on Ca(2+)-regulation and MPT pore opening during simulated ischemia and reperfusion, using fluorescence microscopy. Ventricular cardiomyocytes were isolated from control rat hearts, hearts preconditioned with three cycles of ischemia/reperfusion or naïve myocytes remotely conditioned with effluent collected from preconditioned hearts. Both conventional-IPC and remote-IPC reduced the loss of Ca(2+)-homeostasis and contractile function following reenergization of metabolically inhibited cells and protected myocytes against ischemia/reperfusion injury. However, only conventional-IPC reduced the Ca(2+)-loading during metabolic inhibition and this was independent of any change in sarcKATP channel activity but was associated with a reduction in Na(+)-loading, reflecting a decrease in Na/H exchanger activity. Remote-IPC delayed opening of the MPT pores in response to ROS, which was dependent on PKCε and NOS-signaling. These data show that remote-IPC inhibits MPT pore opening to a similar degree as conventional IPC, however, the contribution of MPT pore inhibition to protection against reperfusion injury is independent of Ca(2+)-loading in remote IPC. We suggest that inhibition of the MPT pore and not Ca(2+)-loading is the common link in cardioprotection between conventional and remote IPC.

Ursolic acid triggers nonprogrammed death (necrosis) in human glioblastoma multiforme DBTRG-05MG cells through MPT pore opening and ATP decline.

SCOPE: Ursolic acid, a natural pentacyclic triterpenic acid, possesses anticancer potential and diverse biological effects, but its correlation with glioblastoma multiforme cells and different modes of cell death is unclear. We studied the cellular actions of human glioblastoma multiforme DBTRG-05MG cells after ursolic acid treatment and explored cell-selective killing effect of necrotic death as a cell fate. METHODS AND RESULTS: Ursolic acid effectively reversed temozolomide resistance and reduced DBTRG-05MG cell viability. Surprisingly, ursolic acid failed to stimulate the apoptosis- and autophagy-related signaling networks. The necrotic death was characterized by annexin V/propidium iodide double-positive detection and release of high-mobility group protein B1 and lactate dehydrogenase. These ursolic acid elicited responses were accompanied by reactive oxygen species generation and glutathione depletion. Rapid mitochondrial dysfunction was paralleled by the preferential induction of necrosis, rather than apoptotic death. Mitochondrial permeability transition (MPT) is a phenomenon to provide the onset of mitochondrial depolarization during cellular necrosis. The opening of MPT pores that were mechanistically regulated by cyclophilin D, and adenosine triphosphate decline occurred in treated necrotic DBTRG-05MG cells. Cyclosporine A (an MPT pore inhibitor) prevented ursolic acid-provoked necrotic death and the acid-involved key regulators. CONCLUSION: Our study is the first to report that ursolic acid-modified mitochondrial function triggers defective death by necrosis in DBTRG-05MG cells rather than augmenting programmed death.

Protective effect of Aloe vera gel on the permeability transition pore in the inner membrane of rat liver mitochondria in vitro.

OBJECTIVE: Aloe vera is a perennial drought resisting, succulent plant belonging to the zanthorrhoeaceae family which historically has been used for a variety of medicinal purposes. This study seeks to determine the effect of varying concentrations of Aloe vera gel (50, 100, 150, 250, and 350 µg/ml) on mitochondrial permeability transition pore (MPTP) in rat liver mitochondria (RLM) (in vitro). METHODS: Fresh Aloe gel was prepared daily from the Aloe vera leaf and the effect of the gel on mitochondrial membrane permeability transition pore opening was estimated in vitro using the spectrophotometric method of Lapidus and Sokolove. RESULTS: Varying concentrations of Aloe vera gel (50, 100, 150, 250, and 350 µg/ml) induced (insignificantly at p < 0.05) the opening of the mitochondrial permeability transition pore in a concentration dependent manner in the absence of calcium (Δ540 nm as -0.020 ± 0.008, -0.021 ± 0.009, -0.031 ± 0.013, -0.031 ± 0.014, -0.034 ± 0.014 respectively) when compared with the control (-0.016 ± 0.009). In the presence of calcium, the various concentrations of Aloe vera gel further opened the MMPT pore with the highest effect noticed at 350 µg/ml concentration. CONCLUSIONS: These findings indicate that Aloe vera gel modulates the mitochondrial pore opening by further increasing the effect of calcium. This effect is needed in situations that requires tissue wastage such as in cancer treatment.