Impact of Swimming Exercise Training on the Effects of Modulation of Mitochondrial Permeability Transition and NOS-1 Activation in Medullary Cardiovascular Neurons of Rats

Physical inactivity can be considered one of the major risk factors related to cardiovascular diseases. There are reasons to believe that the positive effect of exercise training is, to a large extent, mediated by modulation of the nervous control of the circulation system. In our previous studies, we showed that modulation of mitochondrial permeability transition in medullary cardiovascular neurons significantly contributes to the hemodynamic reactions in both the norm and a number of pathological states. In this study, we examined in acute experiments on urethane-anesthetized rats the hemodynamic effects mediated by either modulation of mitochondrial permeability transition in medullary neurons, or activation of neuronal NO synthase (NOS-1) in these neuronal populations after preliminary moderate exercise training (everyday swimming sessions of increased duration carried out for four weeks). It was shown that, after exercise training had been completed, the effects of injections of an inductor of mitochondrial permeability transition pore (MPTP) opening, phenylarsine oxide (PAO, 0.5 to 1.5 nmol), into populations of cardiovascular neurons in the medullary autonomic nuclei (nucl. tractus solitarius and paramedian and lateral reticular nuclei) were less expressed, as compared with those in control (untrained) animals. The data obtained suggest that exercise training can exert a protective action on functional activity of medullary neurons due to the decreased sensitivity of MPTPs to their opening. Injections of an inhibitor of MPTP opening, melatonin (0.7 to 2.1 nmol), into populations of medullary neurons under study in trained rats induced a decrease in the systemic arterial pressure (SAP), in contrast to untrained animals demonstrating mostly hypertensive responses following injections of melatonin into the above nuclei. Injections of an activator of neuronal NO synthase (NOS-1), L-arginine, into the medullary nuclei of swimming-trained rats resulted in more expressed hemodynamic shifts than in control animals, which suggests an increase in the activity of neuronal NO synthase in medullary neurons of such animals.     

Hemodynamic responses induced by modulation of the nitric oxide system and mitochondrial permeability in the medullary cardiovascular nuclei of rats

In acute experiments on normotensive rats and those with genetically determined hypertension (urethane anesthesia), we studied hemodynamic effects resulting from modulation of the activities of neuronal NO synthase (NOS-1), arginase II, and superoxide dismutase, and also of the mitochondrial permeability in medullary cardiovascular neurons. Unilateral microinjections of either a nitric oxide (NO) donor, sodium nitroprusside, or a substrate for endogenous NO synthesis, L-arginine, into the medullary cardiovascular nuclei (nucl. tractus solitarius, NTS, nucl. ambiguous, AMB, paramedian nucleus, PMn, and lateral reticular nucleus LRN) were shown to induce hemodynamic responses with rather similar dynamics in both normotensive and spontaneously hypertensive rats, although in the latter the reactions were more intense. Injections of an antagonist of NOS-1, N^G nitro-L-arginine (L-NNA), into the medullary nuclei under study in spontaneously hypertensive rats resulted in shifts of the systemic arterial pressure (SAP), which did not differ dramatically from those observed in normotensive animals. The data obtained serve as the background for the suggestion that the functional activity of NOS-1 is not fundamentally impaired under hypertension conditions, but, probably, the amount of the substrate for adequate synthesis of NO via the NO-synthase pathway of metabolism of L-arginine is insufficient. Considering this, we examined the functional activity of arginase, an enzyme that also, similarly to NOS, uses L-arginine for metabolic transformation. Injections of antagonists of arginase, norvaline or α-difluoromethylornithine hydrochloride (DFMO), into populations of the medullary neurons under study induced similar shifts of the SAP in normotensive and spontaneously hypertensive rats, and those responses did not differ significantly from the effects of inhibition of the NOS-1 activity. Thus, both the above-mentioned enzymes are potentially active in normotensive and spontaneously hypertensive rats; so, a possibility for their competition for L-arginine in certain situations does exist. Modulation of the mitochondrial permeability in medullary cardiovascular neurons in normotensive and spontaneously hypertensive rats induced significant hemodynamic effects. In particular, an increase in the mitochondrial permeability in the medullary cardiovascular nuclei by injections of an inductor of mitochondrial permeability transition pore (mPTP) opening, phenylarsine oxide (PAO), was accompanied by SAP drops in both normotensive and spontaneously hypertensive rats; the effects were dose-dependent and, in some cases, irreversible. A decrease in the mitochondrial permeability in the neurons under study by injections of an inhibitor of mPTP, melatonin, induced mostly hypertensive responses, although in some experiments we observed hypotensive and two-phase responses. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 232–244, May–June, 2007.     

NADPH-Diaphorase-containing neurons in the medullary structures involved in generation of the respiratory activity in neonatal rats

Using a histochemical technique, we examined distribution of the neurons containing a marker of nitric oxide synthase (NOS), NADPH-diaphorase (NADPH-d), on frontal slices of the medulla and upper cervical spinal segments of 4-day-old rats. It was demonstrated that NADPH-d-positive cells are present within the dorsal and ventral medullary respiratory groups. The highest density of the labeled middle-size multipolar neurons (27.9±2.6 cells per 0.1 mm² of the slice) was observed in the rostral part of the ventral respiratory group, within the reticular lateral paragigantocellular nucleus. Similar NADPH-d-positive neurons were also observed in other reticular formation structures: rostroventrolateral reticular, gigantocellular, and ventral medullary nuclei, and in the ventral part of the paramedial nucleus. There were no labeled neurons in the lateral reticular nucleus. Single small and medium-size labeled neurons were found at all rostro-caudal levels of thenucl. ambiguous (nuclei retrofacialis, ambiguous, andretroam-biguous). Groups of NADPH-d-positive neurons were also revealed within the dorsal respiratory group, along the whole length of thenucl. tractus solitarii (mostly in its ventrolateral parts). Single labeled neurons were also observed in thenucl. n. hypoglossi, and their groups were observed in the dorsal motor part of thenucl. n. vagus. Involvement of the structures containing NADPH-d-positive neurons in the processes related to generation of the respiratory activity is discussed. Our neuroanatomical experiments prove that in early postnatal mammals NO is actively involved in generation and regulation of the medullary respiratory rhythm. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 128–136, March–April, 2000.     

Involvement of Nitric Oxide in the Medullary Control of Circulation in Normotensive Rats

In acute experiments on anesthetized (urethane) normotensive rats, we studied the hemodynamic effects of unilateral microinjections of a nitric oxide (NO) donor, sodium nitroprusside, into the medullary nuclei participating in central cardiovascular control. We studied also the effects of modulation of the intensity of NO production: enhancing its synthesis by intramedullary injections of exogenous L-arginine or inhibiting this process with an inhibitor of neuronal NO synthase (nNOS), L-NNA, or with an inhibitor of arginase, norvaline. Intramedullary injections of the above agents were confined to the nucleus of the tractus solitarius, dorsal motor nucleus of the vagus, nucleus ambiguous, and lateral reticular nucleus. We tried to evaluate the possibility of production of NO from L-arginine in central neurons of normotensive rats via not only the well-known NO synthase pathway, but also via an alternative arginase-mediated pathway of metabolism of the above amino acid. Our results demonstrated that both enzymes are potentially active: injections of the mentioned inhibitors of the enzymes into the medullary neuronal structures induced marked shifts in the systemic arterial pressure (SAP), the integrative parameter characterizing the state of the cardiovascular system. After preliminary administration of an nNOS inhibitor, 7-nitroindazole (30 mg/kg, i.p.) or an inhibitor of arginase, norvaline (2 g, i.v.), injections of L-arginine into the medullary nuclei failed to evoke significant shifts in the SAP. We suggest that the comparative degree of activation of nNOS or arginase in the medullary nuclei depends on different factors, first of all on the level of oxygenation of the nerve tissue. An inverse dependence is likely to exist between the levels of activation of the above enzymes.     

The effect of complexes of precursors and modulators of coenzyme Q biosynthesis on the functional state of heart mitochondria of old rats

Aging is accompanied by changes in activity of electron-transport enzyme complexes in myocardial mitochondria of old rats and by increased sensitivity of the mitochondrial permeability transition pore (MPTP) to inductors of its opening (Ca²⁺ and phenylarsine oxide). We also observed activation of lipid and protein free-radical peroxidation processes. Administration of a complex of biologically active substances that included precursors and modulators of coenzyme Q biosynthesis (α-tocopherol acetate, 4-hydroxybenzoic acid, and methionine) caused the increase in coenzyme Q content, correction of functional activity of mitochondrial electron-transport chain enzyme complexes, the decrease in intensity of lipid and protein free-radical peroxidation in the heart mitochondria and the decrease in sensitivity of mitochondrial permeability transition pore to inductors of its opening. This complex may be recommended for treatment of mitochondrial dysfunction in various pathologies of cardiovascular system, including in aging.     

On the mechanism of palmitic acid-induced apoptosis: the role of a pore induced by palmitic acid and Ca2+ in mitochondria

Palmitic acid (Pal) is known to promote apoptosis (Sparagna G et al (2000) Am J Physiol Heart Circ Physiol 279: H2124–H2132) and its amount in blood and mitochondria increases under some pathological conditions. Yet, the mechanism of the proapoptotic action of Pal has not been elucidated. We present evidence for the involvement of the mitochondrial cyclosporin A-insensitive pore induced by Pal/Ca²⁺ complexes in the apoptotic process. Opening of this pore led to a fall of the mitochondrial membrane potential and the release of the proapoptotic signal cytochrome c. The addition of cytochrome c prevented these effects and recovered membrane potential, which is in contrast to the cyclosporin A-sensitive mitochondrial permeability transition pore. Oleic and linoleic acids prevented the Pal/Ca²⁺-induced pore opening in the intact mitochondria, this directly and significantly correlating with the effect of these fatty acids on Pal-induced apoptosis in cells (Hardy S et al (2003) J Biol Chem 278: 31861–31870). The specific probe for cardiolipin, 10-N-nonyl acridine orange, inhibited formation of this pore.     

The mitochondrial phosphate carrier interacts with cyclophilin D and may play a key role in the permeability transition

The mitochondrial permeability transition pore (MPTP) plays a key role in cell death, yet its molecular identity remains uncertain. Although knock-out studies have confirmed critical roles for both cyclophilin-D (CyP-D) and the adenine nucleotide translocase (ANT), given a strong enough stimulus MPTP opening can occur in the absence of either. Here we provide evidence that the mitochondrial phosphate carrier (PiC) may also be a critical component of the MPTP. Phenylarsine oxide (PAO) was found to activate MPTP opening in the presence of carboxyatractyloside (CAT) that prevents ANT binding to immobilized PAO. Only four proteins from solubilized CAT-treated beef heart inner mitochondrial membranes bound to immobilized PAO, one of which was the PiC. GST-CyP-D pull-down and co-immunoprecipitation studies revealed CsA-sensitive binding of PiC to CyP-D; this increased following diamide treatment. Co-immunoprecipitation of the ANT with the PiC was also observed but was insensitive to CsA treatment. N-ethylmaleimide and ubiquinone analogues (UQ(0) and Ro 68-3400) inhibited phosphate transport into rat liver mitochondria with the same concentration dependence as their inhibition of MPTP opening. UQ(0) and Ro 68-3400 also induced the "m" conformation of the ANT, as does NEM, and reduced the binding of both the PiC and ANT to the PAO column. We propose a model for the MPTP in which a calcium-triggered conformational change of the PiC, facilitated by CyP-D, induces pore opening. An interaction of the PiC with the ANT may enable agents that bind to either transporter to modulate pore opening.     

Central projections of the lagena (the third otolith endorgan of the inner ear) in the pigeon

Central projections of the lagena were studied in the pigeon using transport of biotinylated dextran amine (BDA) that was locally applied to the lagenar epithelium through the opened cochlear canal. Descending (dorsocaudal part) and superior (middle part) vestibular nuclei were the main rhombencephalon structures with the maximum density of labeled fibers and terminals. Lesser numbers of labeled fibers were observed in the ventral part of the lateral vestibular nucleus and also in the medial vestibular nucleus; single labeled fibers were found in the cochlear nuclei. In the cases where BDA diffused not only in the lagena but also on the basilar papilla after application of the marker to the cochlear canal, considerable numbers of labeled fibers were observed in the cochlear nuclei; apart from this, the pattern of distribution of labeled fibers in the vestibular nuclei did not differ in general from that described above (in the case of a sufficiently local application of BDA only to the lagena). Efferent lagenar neurons were localized ventrally with respect to the vestibular nuclei, in particular in the nucl. reticularis pontis caudalis. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 199–210, May–June, 2008.     

One of the Pathways of Transmission of Afferent Activity to the Locus Coeruleus Under Conditions of Immobilization Stress

An electrophysiological proof has been obtained for the morphological data on the existence of afferent inputs from the medullary nucl. praepositus hypoglossi (NPH) to the brainstem locus coeruleus. The crucial role of the NPH in the formation of background spike activity of neurons of the locus coeruleus under conditions of short-term immobilization stress has been demonstrated. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 151–156, March–April, 2005.     

Cytochrome c release precedes mitochondrial membrane potential loss in cerebellar granule neuron apoptosis: lack of mitochondrial swelling

It has been suggested that release of cytochrome c (Cyt c) from mitochondria during apoptotic death is through opening of the mitochondrial permeability transition pore followed by swelling-induced rupture of the mitochondrial outer membrane. However, this remains controversial and may vary with cell type and model system. We determined that in mouse cerebellar granule neurons, Cyt c redistribution preceded the loss of mitochondrial membrane potential during the apoptotic process, suggesting that the pore did not open prior to release. Furthermore, when mitochondria were morphologically assessed by electron microscopy, they were not obviously swollen during the period of Cyt c release. This indicates that the pore mechanism of action, if any, is not through mitochondrial outer membrane rupture. While bongkrekic acid, an inhibitor of pore opening, modestly delayed apoptotic death, it also caused a significant (p < 0.05) suppression of protein synthesis. An equivalent suppression of protein synthesis by cycloheximide had a similar delaying effect, suggesting that bongkrekic acid was acting non-specifically. These findings suggest that mitochondrial permeability transition pore is not involved in Cyt c release from mitochondria during the apoptotic death of cerebellar granule neurons.     

Modulation of the Permeability Transition Pore by Inhibition of the Mitochondrial KATP Channel in Liver vs. Brain Mitochondria

Inhibition of the mitochondrial K_ATP (mitoK_ATP) channel abrogates the beneficial effects of preconditioning induced by a brief episode of sublethal ischemia. We studied the effect of 5-hydroxydecanoate, a well-known inhibitor of the mitoK_ATP channel, on swelling of isolated liver and brain mitochondria. Volume changes were determined by measurement of light absorbance at 540 nm. Mitochondrial swelling induced by adding Ca²⁺ ions correlated with opening of the permeability transition pore as shown by modulation by 1 μM cyclosporin A. In brain mitochondria, 5-hydroxydecanoate did not significantly affect Ca²⁺-induced swelling. In contrast, 50 or 500 μM 5-hydroxydecanoate increased swelling of liver mitochondria by 9.7 ± 5.1% (n = 6, P = 0.057) and 29.4 ± 1.4% (n = 5, P < 0.0001), respectively. The effect of 5-hydroxydecanoate was blocked by cyclosporin A and was dependent on the presence of potassium in the medium. In medium containing 200 μM ATP to inhibit the mitoK_ATP channel, 5–hydroxydecanoate did not further increase Ca²⁺-induced swelling. We conclude that inhibition of the mitoK_ATP channel exerts its detrimental effect by facilitation of permeability transition pore opening.     

Connections of brainstem respiratory nuclei studied by the retrograde horseradish peroxidase axon transport method

Intrabulbar connections of respiratory nuclei and the medullary reticular formation and also descending pathways from these structures in the spinal cord were studied by the retrograde horseradish peroxidase axonal transport method in cats. Neurons of the nucleus ambiguus and nucleus retroambigualis (ventral respiratory group) and of the ventrolateral part of the nucleus of the tractus solitarius (dorsal respiratory group) were shown to form direct two-way connections with each other and with the medial region of the medulla. Neurons of the pneumotaxic center send uncrossed axons to the nucleus ambiguus and to the medial medullary reticular formation. Neurons of the contralateral homonymous nucleus and neurons of the nucleus of the tractus solitarius are sources of projections of the locus coeruleus. A well developed system of direct connections was found between neurons of respiratory nuclei of the two halves of the brain. The possible role of these nuclear formations in genesis of the respiratory rhythm and regulation of the respiratory and other motor functions of the reticular formation is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 2, pp. 149–157, March–April, 1982.     

Medullary reticular neurons in the Japanese toad: morphologies and excitatory inputs from the optic tectum

1. To elucidate the neural mechanisms that mediate visual responses of optic tectum (OT) to medullary and spinal motor systems, we analyzed medullary reticular neurons in paralyzed Japanese toads (Bufo japonicus). We examined their responses to electrical stimulation of OT, and stained some neurons intracellularly. Responses to stimulation of the glossopharyngeal nerve (IX) were also analyzed. 2. Extracellular single unit recording revealed excitatory responses of medullary neurons to OT and IX stimulation. Among 92 units encountered, 79 responded to OT stimuli, 10 to IX stimuli, and 3 to both. Some units responded to successive stimuli of short intervals with relatively stable lags. 3. Intracellular recording and staining experiments revealed morphologies of reticular neurons that received excitatory inputs from OT. Thirteen units were identified after complete reconstruction of somata and dendrites. Neurons in the nucleus reticularis medius received excitatory inputs from bilateral OT. They had wide dendrites in ventral, ventrolateral and lateral funiculi, and single axons descending in the ipsilateral ventral funiculus as far caudally as the cervical spinal cord. Some collaterals of these axons projected directly to the hypoglossal and spinal motor nuclei. Some neurons in other medullary nuclei (nuc. reticularis superior, pretrigeminal nucleus, nuc. reticularis inferior, and nuc. tractus spinalis nervi trigemini) also responded to the OT stimulation. 4. Activities in bilateral OT converge onto medullary reticular neurons, which may directly control medullary and spinal motor systems.     

Topico-anatomical relations among the structures of the caudal medullary region in early postnatal rats

Anatomical and topographical organization of the structures of the caudal medullary region was studied in 3- and 21-day-old rats. A system of stereotaxic coordinates was proposed, and brain maps for the animals of these age groups were plotted. Topographic characteristics were given for the nuclei forming the medullary respiratory center: the lateral reticular nucleus,n. ambiguus, and the nuclei of the solitary tract, vagus and hypoglossal cranial nerves. These structures differ from each other in their cytoarchitectonics and in the changes in number during their neurons' postnatal development. The differences are suggested to result from the differences in morphological maturation of the neurons at early ontogenic stages.Neirofiziologiya/Neurophysiology, Vol. 26, No. 4, pp. 243–250, July–August, 1994.     

Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer's disease

Cyclophilin D (CypD, encoded by Ppif) is an integral part of the mitochondrial permeability transition pore, whose opening leads to cell death. Here we show that interaction of CypD with mitochondrial amyloid-beta protein (Abeta) potentiates mitochondrial, neuronal and synaptic stress. The CypD-deficient cortical mitochondria are resistant to Abeta- and Ca(2+)-induced mitochondrial swelling and permeability transition. Additionally, they have an increased calcium buffering capacity and generate fewer mitochondrial reactive oxygen species. Furthermore, the absence of CypD protects neurons from Abeta- and oxidative stress-induced cell death. Notably, CypD deficiency substantially improves learning and memory and synaptic function in an Alzheimer's disease mouse model and alleviates Abeta-mediated reduction of long-term potentiation. Thus, the CypD-mediated mitochondrial permeability transition pore is directly linked to the cellular and synaptic perturbations observed in the pathogenesis of Alzheimer's disease. Blockade of CypD may be a therapeutic strategy in Alzheimer's disease.     

Role of the mitochondrial membrane permeability transition in cell death

In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received considerable attention. An increase of mitochondrial membrane permeability is one of the key events in apoptotic or necrotic death, although the details of the mechanism involved remain to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca²⁺-dependent increase of mitochondrial membrane permeability that leads to loss of Δψ, mitochondrial swelling, and rupture of the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel that is known as the permeability transition pore (PTP), which putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (Cyp D: a mitochondrial peptidyl prolyl-cis, trans-isomerase), and other molecule(s). Recently, significant progress has been made by studies performed with mice lacking Cyp D at several laboratories, which have convincingly demonstrated that Cyp D is essential for the MPT to occur and that the Cyp D-dependent MPT regulates some forms of necrotic, but not apoptotic, cell death. Cyp D-deficient mice have also been used to show that the Cyp D-dependent MPT plays a crucial role in ischemia/reperfusion injury. The anti-apoptotic proteins Bcl-2 and Bcl-x_L have the ability to block the MPT, and can therefore block MPT-dependent necrosis in addition to their well-established ability to inhibit apoptosis.     

Discrimination between two steps in the mitochondrial permeability transition process

It is well known that a lag phase generally elapses between the addition of inducers of the mitochondrial permeability transition and the opening of the pore. To advance our present understanding as regards the significance of this phenomenon, we used experimental approaches which are sensitive to different aspects of the permeability transition process. The pore conformation was sensed by the fluorescence anisotropy changes of hematoporphyrin-labelled mitochondria. Membrane permeabilization was ascertained by following the matrix swelling consequent to external solute equilibration. We show that the anisotropy changes of mitochondria-bound hematoporphyrin precede both membrane depolarization (proton permeation) and matrix swelling (solute permeation), thus sensing a step of the permeability transition process that involves the pore in its closed state. We suggest that the opening of the pore is preceded by a structural remodelling of mitochondrial domains containing hematoporphyrin-near, pore-regulating histidines. Such a perturbation is strongly inhibited at acidic matrix pH and completely blocked by cyclosporin A. In sucrose-based media the opening of the pore can be strongly delayed, as compared to salt-based media, a fact which probably reflects perturbation of mitochondrial membranes by sugar. We conclude that the mitochondrial permeability transition could be described as an at least two-step process which is mainly regulated by conformational changes of the pore components.     

Neuronal pathways involved in the optokinetic head nystagmus of the frog

The morphology of projection neurons in the basal optic nucleus and the pretectal area and the interconnections of these brain regions were studied with the aid of the cobalt-filling technique. It was found that the nucleus-sends long descending axons to the medullary reticular formation. The two basal optic nuclei are reciprocally interconnected and do not give a direct descending pathway. The pretectal nuclei and the basal optic nucleus are also reciprocally coupled. It is supposed that the described pathways mediate commands for horizontal and vertical nystagmic head movements.     

Hypoxic preconditioning-induced mitochondrial protection is not disrupted in a cell model of mtDNA T8993G mutation-induced F1F0-ATP synthase defect: the role of mitochondrial permeability transition

Transient opening of the mitochondrial permeability transition pore plays a crucial role in hypoxic preconditioning-induced protection. Recently, the cyclophilin-D component of the mitochondrial permeability transition pore has been shown to interact with and regulate the F1F0-ATP synthase. However, the precise role of the F1F0-ATP synthase and the interaction between cyclophilin-D and F1F0-ATP synthase in the mitochondrial permeability transition pore and hypoxic preconditioning remain uncertain. Here we found that a 1-h hypoxic preconditioning delayed apoptosis and improved cell survival after stimulation with various apoptotic inducers including H₂O₂, ionomycin, and arachidonic acid in mitochondrial DNA T8993G mutation (NARP) osteosarcoma 143B cybrids, an F1F0-ATP synthase defect cell model. This hypoxic preconditioning protected NARP cybrid cells against focal laser irradiation-induced oxidative stress by suppressing reactive oxygen species formation and preventing the depletion of cardiolipin. Furthermore, the protective functions of transient opening of the mitochondrial permeability transition pore in both NARP cybrids and wild-type 143B cells can be augmented by hypoxic preconditioning. Disruption of the interaction between cyclophilin-D and F1F0-ATP synthase by cyclosporin A attenuated the mitochondrial protection induced by hypoxic preconditioning in both NARP cybrids and wild-type 143B cells. Our results demonstrate that the interaction between cyclophilin-D and F1F0-ATP synthase is important in the hypoxic preconditioning-induced cell protection. This finding improves our understanding of the mechanism of mitochondrial permeability transition pore opening in cells in response to hypoxic preconditioning, and will be helpful in further developing new pharmacological agents targeting hypoxia–reoxygenation injury and mitochondria-mediated cell death     

Snake phospholipase A2 neurotoxins enter neurons, bind specifically to mitochondria, and open their transition pores

Snake presynaptic neurotoxins with phospholipase A(2) activity are potent inducers of paralysis through inhibition of the neuromuscular junction. These neurotoxins were recently shown to induce exocytosis of synaptic vesicles following the production of lysophospholipids and fatty acids and a sustained influx of Ca(2+) from the medium. Here, we show that these toxins are able to penetrate spinal cord motor neurons and cerebellar granule neurons and selectively bind to mitochondria. As a result of this interaction, mitochondria depolarize and undergo a profound shape change from elongated and spaghetti-like to round and swollen. We show that snake presynaptic phospholipase A(2) neurotoxins facilitate opening of the mitochondrial permeability transition pore, an inner membrane high-conductance channel. The relative potency of the snake neurotoxins was similar for the permeability transition pore opening and for the phospholipid hydrolysis activities, suggesting a causal relationship, which is also supported by the effect of phospholipid hydrolysis products, lysophospholipids and fatty acids, on mitochondrial pore opening. These findings contribute to define the cellular events that lead to intoxication of nerve terminals by these snake neurotoxins and suggest that mitochondrial impairment is an important determinant of their toxicity.