Rat Brain Mitochondria Research Articles

Functional impact of PTP1B-mediated Src regulation on oxidative phosphorylation in rat brain mitochondria.

Given the presence of Src and PTP1B within rat brain mitochondria, we have investigated whether PTP1B regulates Src activity in mitochondria as in the cytosol. Results showed that Src was stimulated by in vitro addition of ATP to mitochondria, and this stimulation was reversed by a membrane-permeable allosteric inhibitor of PTP1B and by a potent selective Src inhibitor. They also indicated a direct action of PTP1B on phosphorylated tyrosine 527 residue of Src, thus implicating a role for PTP1B in the modulation of Src activity in mitochondria. Putative Src and PTP1B substrates were identified by liquid chromatography tandem mass spectrometry and two-dimensional blue native/SDS-PAGE. Both inhibitors inhibited ADP-stimulated respirations concurrently with Src activation and complex IV activation by ATP, while having no effect or increasing the activity of the other complexes. Our analysis emphasizes the regulatory function of Src and its modulation by PTP1B on oxidative phosphorylation in mitochondria.

Electro-pharmacological profile of a mitochondrial inner membrane big-potassium channel from rat brain

Recent studies have indicated a calcium-activated large conductance potassium channel in rat brain mitochondrial inner membrane (mitoBK channel). Accordingly, we have characterized the functional and pharmacological profile of a BK channel from rat brain mitochondria in the present study. Brain mitochondrial inner membrane preparations were subjected to SDS-PAGE analysis and channel protein reconstitution into planar lipid bilayers. Western blotting and antibodies directed against various cellular proteins revealed that mitochondrial inner membrane fractions did not contain specific proteins of the other subcellular compartments except a very small fraction of endoplasmic reticulum. Channel incorporation into planar lipid bilayers revealed a voltage dependent 211 pS potassium channel with a voltage for half activation (V1/2) of 11.4±1.1mV and an effective gating charge zd of 4.7±0.9. Gating and conducting behaviors of this channel were unaffected by the addition of 2.5mM ATP, and 500 nM charybdotoxin (ChTx), but the channel appeared sensitive to 100 nM iberiotoxin (IbTx). Adding 10mM TEA at positive potentials and 10mM 4-AP at negative or positive voltages inhibited the channel activities. These results demonstrate that the mitoBK channel, present in brain mitochondrial inner membrane, displays different pharmacological properties than those classically described for plasma membrane, especially in regard to its sensitivity to iberiotoxin and charybdotoxin sensitivity.

Comparison of superoxide production of rat brain mitochondria analyzed with hydroethidine and MitoSOX

Comparison of superoxide production of rat brain mitochondria analyzed with hydroethidine and MitoSOX

Dicarboxylate carrier-mediated glutathione transport is essential for reactive oxygen species homeostasis and normal respiration in rat brain mitochondria

Glutathione transport into mitochondria is mediated by oxoglutarate (OGC) and dicarboxylate carrier (DIC) in the kidney and liver. However, transport mechanisms in brain mitochondria are unknown. We found that both carriers were expressed in the brain. Using cortical mitochondria incubated with physiological levels of glutathione, we found that butylmalonate, a DIC inhibitor, reduced mitochondrial glutathione to levels similar to those seen in mitochondria incubated without extramitochondrial glutathione (59% of control). In contrast, phenylsuccinate, an OGC inhibitor, had no effect (97% of control). Additional experiments with DIC and OGC short hairpin RNA in neuronal-like PC12 cells resulted in similar findings. Significantly, DIC inhibition resulted in increased reactive oxygen species (ROS) content in and H(2)O(2) release from mitochondria. It also led to decreased membrane potential, increased basal respiration rates, and decreased phosphorus-to-oxygen (P/O) ratios, especially when electron transport was initiated from complex I. Accordingly, we found that DIC inhibition impaired complex I activity, but not those for complexes II and III. This impairment was not associated with dislodgment of complex subunits. These results suggest that DIC is the main glutathione transporter in cortical mitochondria and that DIC-mediated glutathione transport is essential for these mitochondria to maintain ROS homeostasis and normal respiratory functions.

Cis-4-decenoic acid provokes mitochondrial bioenergetic dysfunction in rat brain

Aims In the present work we investigated the in vitro effect of cis-4-decenoic acid, the pathognomonic metabolite of medium-chain acyl-CoA dehydrogenase deficiency, on various parameters of bioenergetic homeostasis in rat brain mitochondria. Main methods Respiratory parameters determined by oxygen consumption were evaluated, as well as membrane potential, NAD(P)H content, swelling and cytochrome c release in mitochondrial preparations from rat brain, using glutamate plus malate or succinate as substrates. The activities of citric acid cycle enzymes were also assessed. Key findings cis-4-decenoic acid markedly increased state 4 respiration, whereas state 3 respiration and the respiratory control ratio were decreased. The ADP/O ratio, the mitochondrial membrane potential, the matrix NAD(P)H levels and aconitase activity were also diminished by cis-4-decenoic acid. These data indicate that this fatty acid acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor. cis-4-decenoic acid also provoked a marked mitochondrial swelling when either KCl or sucrose was used in the incubation medium and also induced cytochrome c release from mitochondria, suggesting a non-selective permeabilization of the inner mitochondrial membrane. Significance It is therefore presumed that impairment of mitochondrial homeostasis provoked by cis-4-decenoic acid may be involved in the brain dysfunction observed in medium-chain acyl-CoA dehydrogenase deficient patients.

Calcium‐induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43

Carbenoxolone (Cbx), a substance from the medicinal licorice is used for anti‐inflammatory treatments. Cbx was already shown to induce opening of the permeability transition pore (PTP) in rat liver mitochondria (RLM). We investigated the mechanism of action of Cbx on Ca2+‐induced PTP opening in rat brain mitochondria (RBM), and in rat heart mitochondria (RHM) and in RLM, in an attempt to identify the molecular target of Cbx in mitochondria. In RBM, RHM and RLM, threshold Ca2+ load induced PTP opening. This was manifested by sudden Ca2+efflux from the mitochondrial matrix, a drop in membrane potential, and swelling of mitochondria. At 1 μM, Cbx enhanced the Ca2+‐induced, Cyclosporine A‐sensitive PTP opening. A well‐known target of Cbx in cells is the connexin family of proteins, which form gap junctions. Moreover, Connexin43 (Cx43) was previously found in RHM and attributed to the protective mechanism induced by ischemic preconditioning. We hypothesized that Cx43 might be a target for Cbx in brain mitochondria. We clearly detected Cx43 by Western blot in RBM and in mitochondria isolated from astrocytes, less in RHM, but not in RLM. Interestingly, the anti‐Cx43 antibody abolished the Cbx‐enhanced PTP opening in RBM, but not in RLM. In RBM, PTP caused serine‐368 phosphorylation of Cx43. These results show that Cbx facilitates Ca2+‐induced PTP opening, addressing as possible molecular target Cx43 in brain mitochondria.

Protective effects of Aloe vera extract on mitochondriaof neuronal cells and rat brain

To investigate the effects of Aloe vera extract (AV) on mitochondria in rat pheochromocytoma (PC12) cells and rat brain and to study the mechanism of its neuroprotection. After treatment, the morphology of PC12 cells was observed under microscope, the activity of mitochondria in PC12 cells was measured by MTT method, and the mitochondrial membrane potential (MMP) in PC12 cells was detected by JC-1 method. The mitochondrial function in rat brain was detected by resazurin method. The production of malondialdehyd (MDA) in rat brain mitochondria was tested by thiobarbaturic acid (TBA) assay. AV could improve mitochondrial damage induced by azide sodium (NaN3) in PC12 cells. The viability of PC12 cells treated with NaN364 mmol x L(-1) for 4 h decreased by 47.8%, and AV at 1 and 10 mg x L(-1) could respectively increase the viability of NaN3-treated cells by 16.7% (P < 0.05) and 22.3% (P < 0.01). MMP in PC12 cells in AV 1 and 10 mg x L(-1) group was significantly higher than that of NaN3-treated group (P < 0.05). AV also protected the structure and function of mitochondria in rat brain. AV at 10 mg x L(-1) had protective effect on mitochondria function impair induced by NaN3 (P < 0.01). AV 1 and 10 mg x L(-1) markedly inhibited the lipid peroxidation of brain mitochondria induced by Fe2+ -cysteine (P < 0.05, P < 0.01). AV has protective effects on mitochondria of neuronal cells and rat brain.

Functional state of rat brain mitochondria at hypoglycemia convulsive syndrome and different ways of its arresting

Respiratory and phosphorylation functions of rat brain mitochondria was studied under conditions insulin shock and after its treatment with glucose or glutamate (in combination with inhalation of hypercapnic gas mixture--air enriched with 7% CO2). Certain differences in the effects of the applied agents were found. Phosphorylation ability of mitochondria did not reach the normal level even one day after both ways of convulsive state treatment. Some respiratory parameters suggest that unfavorable changes in the respiratory chain functioning mainly occur at the respiratory chain complex I.

Agmatine prevents the Ca2+-dependent induction of permeability transition in rat brain mitochondria

The arginine metabolite agmatine is able to protect brain mitochondria against the drop in energy capacity by the Ca(2+)-dependent induction of permeability transition (MPT) in rat brain mitochondria. At normal levels, the amine maintains the respiratory control index and ADP/O ratio and prevents mitochondrial colloid-osmotic swelling and any electrical potential (DeltaPsi) drop. MPT is due to oxidative stress induced by the interaction of Ca(2+) with the mitochondrial membrane, leading to the production of hydrogen peroxide and, subsequently, other reactive oxygen species (ROS) such as hydroxyl radicals. This production of ROS induces oxidation of sulfhydryl groups, in particular those of two critical cysteines, most probably located on adenine nucleotide translocase, and also oxidation of pyridine nucleotides, resulting in transition pore opening. The protective effect of agmatine is attributable to a scavenging effect on the most toxic ROS, i.e., the hydroxyl radical, thus preventing oxidative stress and consequent bioenergetic collapse.

Agmatine transport in brain mitochondria: a different mechanism from that in liver mitochondria

The diamine agmatine (AGM), exhibiting two positive charges at physiological pH, is transported into rat brain mitochondria (RBM) by an electrophoretic mechanism, requiring high membrane potential values and exhibiting a marked non-ohmic force-flux relationship. The mechanism of this transport apparently resembles that observed in rat liver mitochondria (RLM), but there are several characteristics that strongly suggest the presence of a different transporter of agmatine in RBM. In this type of mitochondria, the extent of initial binding and total accumulation is higher and lower, respectively, than that in liver; saturation kinetics and the flux-voltage relationship also exhibit different trends, whereas idazoxan and putrescine, ineffective in RLM, act as inhibitors. The characteristics of agmatine uptake in RBM lead to the conclusion that its transporter is a channel with two asymmetric energy barriers, showing some characteristics similar to those of the imidazoline receptor I(2) and the sharing with the polyamine transporter.

Reactive oxygen species (ROS) in the human neocortex: Role of aging and cognition

Reactive oxygen species (ROS), formed during normal aerobic metabolism, are involved in signal transduction and cognitive functions, but highly increased ROS concentrations may also have detrimental effects. The aim of the present study was to investigate whether aging and cognitive functions are associated with ROS generation in human neocortex obtained from neurosurgical patients. ROS formation in mitochondria from fresh and re-thawed neocortical specimens was measured by monitoring ROS-mediated conversion of dihydrorhodamine 123 to fluorescent rhodamine 123. The validity of this technique was characterized in rat brain mitochondria. The increase in the concentration–response curve of the complex I inhibitor rotenone on ROS generation, as measured by rhodamine 123 (Rh123) fluorescence, was much more pronounced than that of rotenone on mitochondrial [ 3H]-choline uptake [which indicates changes in the mitochondrial membrane potential (Δ Ψ M)]. Thus, mitochondrial ROS generation can be shown by Rh123 fluorescence although this fluorescence may also reflect changes in Δ Ψ M to some extent. ROS formation in human brain mitochondria positively correlated with the age of patients. Moreover, an age-corrected positive correlation of ROS formation with presurgical cognitive performance was observed. Our data suggest a mild increase in ROS formation with aging possibly reflecting a physiological compensation of mitochondrial function. Furthermore, higher cognitive performances in tests of executive functions may be paralleled by slightly increased ROS levels.

Protection against malathion-induced oxidative stress in rat brain mitochondria by phosphodiesterase (PDE) type 4 selective inhibitor

Protection against malathion-induced oxidative stress in rat brain mitochondria by phosphodiesterase (PDE) type 4 selective inhibitor

Single channel studies of the ATP-regulated potassium channel in brain mitochondria

Mitochondrial potassium channels in the brain have been suggested to have an important role in neuroprotection. The single channel activity of mitochondrial potassium channels was measured after reconstitution of the purified inner membrane from rat brain mitochondria into a planar lipid bilayer. In addition to a large conductance potassium channel that was described previously, we identified a potassium channel that has a mean conductance of 219 +/- 15 pS. The activity of this channel was inhibited by ATP/Mg(2+) and activated by the potassium channel opener BMS191095. Channel activity was not influenced either by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-regulated potassium channels, or by the plasma membrane ATP-regulated potassium channel blocker HMR1098. Likewise, this mitochondrial potassium channel was unaffected by the large conductance potassium channel inhibitor iberiotoxin or by the voltage-dependent potassium channel inhibitor margatoxin. The amplitude of the conductance was lowered by magnesium ions, but the opening ability was unaffected. Immunological studies identified the Kir6.1 channel subunit in the inner membrane from rat brain mitochondria. Taken together, our results demonstrate for the first time the single channel activity and properties of an ATP-regulated potassium channel from rat brain mitochondria.

Biapigenin Modulates the Activity of the Adenine Nucleotide Translocator in Isolated Rat Brain Mitochondria

In this study, we investigated the effects of biapigenin, a biflavone present in the extracts of Hypericum perforatum, in rat brain mitochondrial bioenergetics and calcium homeostasis. We found that biapigenin significantly decreased adenosine diphosphate (ADP)-induced membrane depolarization and increased repolarization (by 68 and 37%, respectively). These effects were blocked by atractyloside and bongkrekic acid, but not oligomycin. In the presence of biapigenin, an ADP-stimulated state 3 respiration was still noticeable, which did not happen in the presence of adenine nucleotide translocator (ANT) inhibitors. Taking in consideration the relevance of the ANT in the modulation of the mitochondrial permeability transition pore (mPTP), mitochondrial calcium homeostasis was evaluated alone or in the presence of biapigenin. We found that biapigenin reduces mitochondrial calcium retention by increasing calcium efflux, an effect that was blocked by ADP plus oligomycin, an efficient blocker of the mPTP in brain mitochondria. Taken together, the results in this article suggest that biapigenin modulates mPTP opening, possibly by modulating ANT function, contributing for enhanced mitochondrial calcium efflux, thereby reducing calcium burden and contributing for neuroprotection against excitotoxicity.

A sensitive assay for ornithine amino transferase in rat brain mitochondria by ninhydrin method

To establish/develop an assay method for measuring Ornithine Aminotransferase (EC.2.6.1.13) activity using rat brain mitochondria as a source of enzyme in presence and absence of Pyridoxal Phosphate (PLP). The modified method, with the improved sensitivity, is adopted for the assay of ornithine amino transferase activity in rat brain mitochondria. The enzyme activity was measured at 620 nm, the study showed that reaction was optimum at 37°C for 30 minutes. The assay is sensitive enough to detect activity at the order of nanomoles pyrroline-5-carboxylate/mg protein/minute and can be compared as an alternative to the radio isotopic method which is more cumbersome and aminobenzaldehyde method which is less sensitive. The K(m) & V(max) shows maximum activity in the presence of Pyridoxal Phosphate (Coenzyme) concentration at 0.05mM when compared with absence of Pyridoxal Phosphate as higher the concentration of Pyridoxal Phosphate affects the affinity of the enzyme to substrate. The OAT activity in different tissues of the rat was also studied and highest activity was found in liver and kidney.

Brain monoamine oxidase A in hyperammonemia is regulated by NMDA receptors

AbstractMitochondrial enzyme monoamine oxidase A (MAO-A) generates hydrogen peroxide (H2O2) and is up-regulated by Ca2+ and presumably by ammonia. We hypothesized that MAO-A may be under the control of NMDA receptors in hyperammonemia. In this work, the in vivo effects of single dosing with ammonia and NMDA receptor antagonist MK-801 and the in vitro effect of Ca2+ on MAO-A activity in isolated rat brain mitochondria were studied employing enzymatic procedure. Intraperitoneal injection of rats with ammonia led to an increase in MAO-A activity in mitochondria indicating excessive H2O2 generation. Calcium added to isolated mitochondria stimulated MAO-A activity by as much as 84%. MK-801 prevented the in vivo effect of ammonia, implying that MAO-A activation in hyperammonemia is mediated by NMDA receptors. These data support the conclusion that brain mitochondrial MAO-A is regulated by the function of NMDA receptors. The enzyme can contribute to the oxidative stress associated with hyperammonemic conditions such as encephalopathy and Alzheimer’s disease. The attenuation of the oxidative stress highlights MAO-A inactivation and NMDA receptor antagonists as sources of novel avenues in the treatment of mental disorders.

Effect of moderate hypoxia/reoxygenation on mitochondrial adaptation to acute severe hypoxia

In an experimental model, it was shown that repetitive periods of hypoxia/reoxygenation (H/R) [5 cycles of 5 min hypoxia (12% O(2) in N(2) ) followed by 15 min normoxia, daily for three weeks] attenuated basal and stimulated in vitro lipid peroxidation, as well as H(2)O(2) production in liver and brain mitochondria of rats exposed to acute severe hypoxia. Adaptation to moderate H/R enhanced in mitochondria the production and activity of reactive oxygen species scavengers, such as glutathione, manganese superoxide dismutase, glutathione peroxidase, and glutathione-S-transferase. It was demonstrated that the maintenance of GSH-redox cycle by activation of glutathione reductase and NADP(+) -dependent isocitrate dehydrogenase is an integral part of the biochemical adaptive mechanism of oxidative tolerance to new damaging factor. Brain mitochondria showed more sensitivity to oxidative stress than liver mitochondria, and long-lasting sessions of H/R affect differentially their pro-/antioxidant homeostasis.

The brain‐specific protein, p42IP4(ADAP 1) is localized in mitochondria and involved in regulation of mitochondrial Ca2+

In brain, p42(IP4) (centaurin-alpha1; recently named ADAP 1, which signifies ADP ribosylation factor GTPase activating protein with dual PH domains 1, within the large family of Arf-GTPase activating proteins) is mainly expressed in neurons. p42(IP4) operates as a dual receptor recognising two second messengers, the soluble inositol(1,3,4,5)tetrakisphosphate and the lipid phosphatidylinositol(3,4,5)trisphosphate. We show here for the first time that p42(IP4) is localized in mitochondria, isolated from rat brain and from cells transfected with p42(IP4). In rat brain mitochondria we additionally found interaction of p42(IP4) with 2', 3'-cyclic nucleotide 3'-phosphodiesterase and alpha-tubulin by pull-down binding assay and by immunoprecipitation. In mitochondria from Chinese hamster ovary cells, p42(IP4) is predominantly associated with the intermembrane space and the inner membrane. This localization of p42(IP4) indicates that p42(IP4) might have a still unknown mitochondrial function. We studied whether p42(IP4) is involved in Ca(2+)-induced permeability transition pore opening, which is important in mitochondrial events leading to programmed cell death. We used mouse neuroblastoma cells as a model for the functional studies of p42(IP4) in mitochondria. In mitochondria isolated from p42(IP4)-transfected mouse neuroblastoma cells, over-expression of p42(IP4) significantly decreased Ca(2+) capacity and lag time for Ca(2+) retention. Thus, we suggest that p42(IP4) is involved in the regulation of Ca(2+) transport in mitochondria. We propose that p42(IP4) promotes Ca(2+)-induced permeability transition pore opening and thus destabilizes mitochondria.

The study of ubiquitin-dependent increase in monoamine oxidase sensitivity to proteolysis and specific inhibitor, pargyline

Insertion of exogenous ubiquitin into rat brain mitochondria in the presence of ATP and the ATPregenerating system (creatine phosphate/creatine phosphokinase) results in the increase in: sensitivity of mitochondrial monoamine oxidases (MAO) A and B to inhibition by mechanism based inhibitor and incorporation of [3H]-pargyline, which was especially notable in the fraction obtained by immunoprecipitation of mitochondrial proteins with anti-ubiquitin antiserum and protein A Sepharose. This suggests that MAO is a potential substrate for ubiquitination in vitro. However, the content of the tritium label in this fraction was less than 0.1 % and not more than 0.25% of total radioactivity of [3H]-pargyline bound to control and ATP-ubiquitin treated mitochondria, respectively. Insertion of ubiquitin into mitochondria did not influence molecular masses of [3H]-pargyline labeled proteins. These results suggest that direct ubiquitination of MAO insignificantly contributes to marked changes in the sensitivity of MAO A and MAO B to proteolysis and specific inhibition found under these experimental conditions. It is possible that more complex processes are involved into realization of these effects during ATP-dependent ubiquitin incorporation into mitochondria.

The Neuromediator Glutamate, through Specific Substrate Interactions, Enhances Mitochondrial ATP Production and Reactive Oxygen Species Generation in Nonsynaptic Brain Mitochondria

The finding that upon neuronal activation glutamate is transported postsynaptically from synaptic clefts and increased lactate availability for neurons suggest that brain mitochondria (BM) utilize a mixture of substrates, namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We studied how glutamate affected oxidative phosphorylation and reactive oxygen species (ROS) production in rat BM oxidizing pyruvate + malate or succinate. Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM oxidizing glutamate + malate. Up to 70% of ROS generation was associated with reverse electron transport. These effects of pyruvate + glutamate + malate were observed only with BM and not with liver or heart mitochondria. The effects of glutamate + pyruvate on succinate-supported respiration and ROS generation were not organ-specific and depended only on whether mitochondria were isolated with or without bovine serum albumin. With the non-bovine serum albumin brain and heart mitochondria oxidizing succinate, the addition of pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We conclude that (i) during neuronal activation, simultaneous oxidation of glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an inherent mechanism that protects against ROS generation during reverse electron transport.