mPT Opening Research Articles

Comparative antimalarial, toxicity and mito-protective effects of Diospyros mespiliformis Hochst. ex A. DC. and Mondia whitei (Hook.f.) Skeels on Plasmodium berghei infection in mice

Ethnopharmacological relevanceDiospyros mespiliformis Hochst. ex A. DC. and Mondia whitei (Hook.f.) Skeels are traditionally used in Africa for the treatment of malaria. However, scientific evidence to substantiate this folkloric claim and their effects on liver mitochondria during malaria treatment have not been reported. Aim of the studyThis study investigated the efficacy of D. mespiliformis and M. whitei against chloroquine-sensitive and resistant strains of malarial parasites in mice. It also investigated the toxicity and protection against cellular organelles like mitochondria. Materials and methodsMale Swiss mice were infected with a chloroquine resistant (ANKA) strain of Plasmodium berghei and were treated via oral gavage with methanol extracts of D. mespiliformis and M. whitei reconstituted in diluted dimethylsulfoxide as vehicle (DMSO, 5% v/v) for five consecutive days. Percentage parasite load and clearance were assessed by microscopy. The infected control was treated with the vehicle. Hematological indices were assessed using standard procedures. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined using assay kits. Hepatic mitochondria were isolated via centrifugation, and their permeability transition (mPT), ATPase (mATPase) activity and lipid peroxidation (mLPO) were determined spectroscopically. Liver tissue histology was carried out by standard laboratory procedures. Phytochemical analysis of both extracts were performed using LC-MS to identify the most prominent compounds from each of the extracts. ResultsAfter treatment on day 5, D. mespiliformis and M. whitei at 400 mg/kg decreased mean values for: percentage parasitemia (5.0 ± 1.0, 2.0 ± 0.2), increased Packed Cell Volume (PCV) (36.0 ± 1.4, 36.0 ± 0.0%) and platelets (2.0 ± 1.4, 2.0 ± 2.8 × 105mm3) relative to the untreated control (20.0 ± 5.2; 30.0 ± 0.0%; 1.4 ± 1.4 × 105 mm3, respectively). At the same dose, D. mespiliformis and M. whitei decreased ALT (8.0 ± 3.8, 24.2 ± 4.0U/L), AST (6.2 ± 0.8, 8.0 ± 0.9U/L) and ALP (56.0 ± 0.7, 51.0 ± 1.0U/L) activities compared to the infected control (77.0 ± 10.9U/L, 14.0 ± 0.7U/L and 76.0 ± 6.0U/L, respectively). Both D. mespiliformis and M. whitei reversed mPT opening, decreased mATPase enhancement and mLPO, relative to the control. Histopathology of the liver showed extensive hemorrhagic lesions and severe disseminated congestion in the infected control while both D. mespiliformis and M. whitei were well tolerated at the highest dose. The LC-MS analysis of D. mespiliformis showed the presence of betulinic acid, tocopherol and kaempferol with antimalarial and antioxidant properties while the M. whitei sample contained coumarin and chlorogenic acid that have antimalarial and hepato-protective properties. ConclusionsD.mespiliformis and M. whitei show antimalarial effects against resistant Plasmodium berghei infection, enhanced cell viability, mito-protection and are not toxic in mice.

Abstract 429: Assessment of Functional Mitochondrial Parameters in Single Cardiomyocytes Reveals Critical Events Not Identified in Cell Populations

In cardiomyocytes (CMs), mitochondria play a dual role, maintaining the high energy supply required for rhythmic contraction, but also regulating critical cell death signaling. Impaired mitochondrial function can affect cellular homeostasis and contribute to sub-lethal injury; if mitochondrial impairment is more severe or persistent, pro-death pathways are activated. It is becoming increasingly clear, however, that within a population of cells there is considerable heterogeneity in mitochondrial function between individual CMs; and within a single cell there is also heterogeneity between individual mitochondria/mitochondrial regions. We have developed a high-throughput fluorescence imaging platform to quantitate single CM mitochondrial function in large numbers of cells, yielding information missed using standard assays that evaluate cell populations. When CMs are exposed to H 2 O 2 , they exhibit a dramatic hyperpolarization prior to the loss of mitochondrial membrane potential at the onset of cell contraction and death. There is marked heterogeneity in the timing of this response, with three distinct populations identified using Gaussian mixture models, and the duration of hyperpolarization longer for CMs that hypercontract earlier after H 2 O 2 exposure. This hyperpolarization is accompanied by a simultaneous increase in [Ca 2+ ] i and is preceded by an increase in ROS. Standard methods, which average populations of cells, miss these responses. Blockade of MPT opening with cyclosporine A delays hypercontracture and cell death but does not prevent hyperpolarization. Finally, we have used our platform to track individual mitochondria/mitochondrial regions within a single live CM, identifying mitochondrial heterogeneity within a single cell, which is increased after exposure to H 2 O 2 , isoproterenol or in CMs from mice after ischemia-reperfusion injury. By tracking individual CMs and individual mitochondria within a single CM, we have opened a window to the complex heterogeneities in mitochondrial stress response.

Alterations in voltage-sensing of the mitochondrial permeability transition pore in ANT1-deficient cells

The probability of mitochondrial permeability transition (mPT) pore opening is inversely related to the magnitude of the proton electrochemical gradient. The module conferring sensitivity of the pore to this gradient has not been identified. We investigated mPT’s voltage-sensing properties elicited by calcimycin or H2O2 in human fibroblasts exhibiting partial or complete lack of ANT1 and in C2C12 myotubes with knocked-down ANT1 expression. mPT onset was assessed by measuring in situ mitochondrial volume using the ‘thinness ratio’ and the ‘cobalt-calcein’ technique. De-energization hastened calcimycin-induced swelling in control and partially-expressing ANT1 fibroblasts, but not in cells lacking ANT1, despite greater losses of mitochondrial membrane potential. Matrix Ca2+ levels measured by X-rhod-1 or mitochondrially-targeted ratiometric biosensor 4mtD3cpv, or ADP-ATP exchange rates did not differ among cell types. ANT1-null fibroblasts were also resistant to H2O2-induced mitochondrial swelling. Permeabilized C2C12 myotubes with knocked-down ANT1 exhibited higher calcium uptake capacity and voltage-thresholds of mPT opening inferred from cytochrome c release, but intact cells showed no differences in calcimycin-induced onset of mPT, irrespective of energization and ANT1 expression, albeit the number of cells undergoing mPT increased less significantly upon chemically-induced hypoxia than control cells. We conclude that ANT1 confers sensitivity of the pore to the electrochemical gradient.

Mitochondrial alterations during Al-induced PCD in peanut root tips

Previous study found there was a negative relationship between Al-induced PCD and Al-resistance in peanut. The present research was undertaken to verify whether mitochondria play a significant role in PCD induced by Al in peanut. The roots of Al-tolerant plants were found to exhibit more intensive root growth, while accumulating less Al3+ than Al-sensitive plants under Al treatment. The different enhancement of ROS production was observed in the mitochondria isolated from two peanut cultivars. The concentration of mitochondrial MDA in root tips increased after Al treatment, which was higher in Zhonghua 2 than in 99-1507. With the increase of Al concentration, mitochondrial Ca2+ concentration decreased, and Ca2+ concentration of Zhonghua 2 decreased faster than that of 99-1507. The opening of mitochondrial permeability transition pore was more extensively in mitochondria isolated from Zhonghua 2 than from 99-1507. The collapse of inner mitochondrial membrane potential (ΔΨm) was also observed with a release of Cytochrome c (Cyt c) from mitochondria, it was more obvious in Zhonghua 2 than in 99-1507 with Al concentration increasing. The results showed that mitochondrial membrane structure and function were damaged seriously in Al-induced PCD, the increase of mitochondrial antioxidant system activity decreased cellular damages under Al stress. To sum up, compared with Al-sensitive peanut cultivar, Al-tolerant peanut cultivar has less Al3+ absorption, mitochondrial ROS and membrane lipid peroxidation level, higher control of MPT opening, ΔΨm maintaining, Cty c release from mitochondria and mitochondrial respiratory functions so that it is not easy to produce PCD under Al stress.

Interactions of Cr(VI) and Cr(III) with isolated rat liver mitochondria

article The mechanism of interaction of Cr(VI) with isolated rat liver mitochondria was investigated in this study. The results suggest that Cr(VI) induces the opening of the membrane permeability transition pore (MPT). The phenomenon is cyclosporine-sensitive and is in agreement with the cyclosporine-sensitive apoptosis observed in the cells incubated with this compound. Moreover the action of Cr(III), that is formed in the cells by a reduction of Cr(VI), has been also analysed. The results obtained demonstrated that the Cr(III) does not induce the opening of the MPT in isolated mitochondria, but it has a protective effect in preventing Cr(VI) MPT opening. Therefore, these results suggest that apoptosis is regulated by a balance between Cr(VI) accumulation in the cytoplasm and Cr(III) formation.

Measurements of mitochondrial pH in cultured cortical neurons clarify contribution of mitochondrial pore to the mechanism of glutamate-induced delayed Ca 2+ deregulation

To clarify the role of the mitochondrial permeability transition pore (MPT) in the mechanism of the glutamate-induced delayed calcium deregulation (DCD) and mitochondrial depolarization (MD), we studied changes in cytosolic (pH c) and mitochondrial pH (pH m) induced by glutamate in cultured cortical neurons expressing pH-sensitive fluorescent proteins. We found that DCD and MD were associated with a prominent pH m decrease which presumably resulted from MPT opening. This pH m decrease occurred with some delay after the onset of DCD and MD. This argued against the hypothesis that MPT opening plays a dominant role in triggering of DCD. This conclusion was also supported by experiments in which Ca 2+ was replaced with antagonist of MPT opening Sr 2+. We found that in Sr 2+-containing medium glutamate-induced delayed strontium deregulation (DSD), similar to DCD, which was accompanied by a profound MD. Analysis of the changes in pH c and pH m associated with DSD led us to conclude that MD in Sr 2+-containing medium occurred without involvement of the pore. In contrast, in Ca 2+-containing medium such “non-pore mechanism” was responsible only for MD initiation while in the final stages of MD development the MPT played a major role.

EXERCISE ATTENUATES CALCIUM-TRIGGERED MITOCHONDRIAL PERMEABILITY TRANSITION IN SKELETAL MUSCLE MITOCHONDRIA

An event now recognized to be important in ontogenesis of both muscle cells and mitochondria (mit.) is the mit. permeability transition (MPT). MPT represents an abrupt increase of permeability of the mit. inner membrane following Ca2+ uptake. Exercise is associated with changes in intracellular milieu that may promote MPT pore assembly (e.g. increase in Ca2+ and Pi, oxidative stress). PURPOSE To investigate the effect of single bout of prolonged exercise on the capacity of muscle mit. to sequester Ca2+ and resist MPT. METHODS 7 subjects performed 75 min of cycling at 70% of VO2peak. Mit. were isolated from biopsies taken from the lateral aspect of the m. quadriceps femoris before, immediately after and 2 h after the exercise. Mit. swelling was monitored as changes in A540. Ca2+ pulses (2 μmol of Ca2+/U citrate synthase (CS)) were added every 3 min to the mit. energized with pyruvate+malate until rapid swelling of mit. indicating MPT opening was induced. RESULTS Mit. from samples taken prior to exercise buffered 8.6±0.8 μmol of Ca2+/U CS before they had been saturated and MPT pore was opened. Mit. from samples taken immediately after exercise had a significantly better capacity to sequester repeated Ca2+ loads (13.2±1.2 μmol Ca2+/U CS, P <0.01 vs pre-exercise value). After 2 h recovery the amount of Ca2+ required to elicit progressive swelling of isolated mit. (10.0±0.8 μmol Ca2+/U CS) was not significantly different from that prior to exercise. CONCLUSION Prolonged exercise induces a reversible increase in mit. capacity to accumulate Ca2+ and resist MPT. This effect of exercise resembles protective effect of calcium- or ischemia preconditioning that is known to inhibit MPT and preserve mit. function. MPT opening is a prerequisite for degradation of mit. by the process of autophagy. Improved mit. MPT-resistance induced by prolonged exercise may decrease the rate of degradation of muscle mit. and contribute, along with increased rate of mit. biogenesis, to expansion of muscle mit. mass induced by endurance training.

Mitochondria – the kraken wakes!

Mitochondria – the kraken wakes!