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Biomedical subjects

Alicia J Kowaltowski

Publications and source records attributed to Alicia J Kowaltowski.

At least 19 recordsLinked to original sources
PubMed2006

Pharmacological and physiological stimuli do not promote Ca(2+)-sensitive K+ channel activity in isolated heart mitochondria.

OBJECTIVE: Mitochondrial calcium-activated K(+) (mitoK(Ca)) channels have been described as channels that are activated by Ca(2+), inner mitochondrial membrane depolarization and drugs such as NS-1619. NS-1619 is cardioprotective, leading to the assumption that this effect is related to the opening of mitoK(Ca) channels. Here, we show several weaknesses in this hypothesis. METHODS: Isolated mitochondria from rat hearts were tested for evidence of mitoK(Ca) activity by analyzing functional parameters in K(+)-rich and K(+)-free media. RESULTS: NS-1619 promoted mitochondrial depolarization both in K(+)-rich and K(+)-free media. Respiratory rate increments were also seen in the presence of NS-1619 for both media. In parallel, NS-1619 promoted respiratory inhibition, as evidenced by respiratory measurements in state 3. Mitochondrial volume measurements conducted using light scattering showed that NS-1619 led to swelling, in a manner unaltered by inhibitors of mitoK(Ca) channels, antagonists of adenosine triphosphate-sensitive potassium channels or inhibitors of the permeability transition. Swelling was also maintained when K(+) in the media was substituted with tetraethylammonium (TEA(+)), which is not transported by any known K(+) carrier. Electron microscopy experiments gave support to the idea that NS-1619-induced mitochondrial swelling took place in the absence of K(+). In addition to testing the pharmacological effects of NS-1619, we attempted, unsuccessfully, to promote mitoK(Ca) activity by altering Ca(2+) concentrations in the medium and inducing mitochondrial uncoupling. CONCLUSION: Our data indicate that NS-1619 promotes non-selective permeabilization of the inner mitochondrial membrane to ions, in addition to partial respiratory inhibition. Furthermore, we found no specific K(+) transport in isolated heart mitochondria compatible with mitoK(Ca) opening, whether by pharmacological or physiological stimuli. Our results indicate that NS-1619 has extensive mitochondrial effects unrelated to mitoK(Ca) and suggest that tissue protection mediated by NS-1619 may occur through mechanisms other than activation of these channels.

Animals↗
PubMed2006

Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging.

Replicative life span in Saccharomyces cerevisiae is increased by glucose (Glc) limitation [calorie restriction (CR)] and by augmented NAD+. Increased survival promoted by CR was attributed previously to the NAD+-dependent histone deacetylase activity of sirtuin family protein Sir2p but not to changes in redox state. Here we show that strains defective in NAD+ synthesis and salvage pathways (pnc1delta, npt1delta, and bna6delta) exhibit decreased oxygen consumption and increased mitochondrial H2O2 release, reversed over time by CR. These null mutant strains also present decreased chronological longevity in a manner rescued by CR. Furthermore, we observed that changes in mitochondrial H2O2 release alter cellular redox state, as attested by measurements of total, oxidized, and reduced glutathione. Surprisingly, our results indicate that matrix-soluble dihydrolipoyl-dehydrogenases are an important source of CR-preventable mitochondrial reactive oxygen species (ROS). Indeed, deletion of the LPD1 gene prevented oxidative stress in npt1delta and bna6delta mutants. Furthermore, pyruvate and alpha-ketoglutarate, substrates for dihydrolipoyl dehydrogenase-containing enzymes, promoted pronounced reactive oxygen release in permeabilized wild-type mitochondria. Altogether, these results substantiate the concept that mitochondrial ROS can be limited by caloric restriction and play an important role in S. cerevisiae senescence. Furthermore, these findings uncover dihydrolipoyl dehydrogenase as an important and novel source of ROS leading to life span limitation.

Base Sequence↗
PubMed2006

Inhibition of specific electron transport pathways leads to oxidative stress and decreased Candida albicans proliferation.

Candida parapsilosis mitochondria contain three respiratory chains: the classical respiratory chain (CRC), a secondary parallel chain (PAR) and an "alternative" oxidative pathway (AOX). We report here the existence of similar pathways in C. albicans. To observe the capacity of each pathway to sustain yeast growth, C. albicans cells were cultured in the presence of inhibitors of these pathways. Antimycin A and KCN totally abrogated yeast growth, while rotenone did not prevent proliferation. Furthermore, rotenone promoted only partial respiratory inhibition. Lower concentrations of KCN that promote partial inhibition of respiration did not inhibit yeast growth, while partial inhibition of respiration with antimycin A did. Similarly, AOX inhibitor BHAM decreased O(2) consumption slightly but completely stunted cell growth. Reactive oxygen species production and oxidized glutathione levels were enhanced in cells treated with antimycin A or BHAM, but not rotenone or KCN. These findings suggest that oxidative stress prevents C. albicans growth.

Antimycin A↗
PubMed2006

Mitochondrial Ca2+ transport, permeability transition and oxidative stress in cell death: implications in cardiotoxicity, neurodegeneration and dyslipidemias.

Mitochondrial Ca2+ transport is important in the maintenance of intracellular ion homeostasis, and also a key factor in the pathogenesis of many diseases. We discuss here the main aspects of mitochondrial Ca2+ transport, and how this transport is linked to changes in energy metabolism and redox state. Mitochondrial permeability transition, a consequence of excessive mitochondrial Ca2+ accumulation associated with oxidative stress is also discussed. Finally, our current understanding of the involvement of these mitochondrial processes in cardiac ischemia-reperfusion, neurodegeneration and dyslipidemias is presented.

Biological Transport↗
PubMed2006

Hyperlipidemic mice present enhanced catabolism and higher mitochondrial ATP-sensitive K+ channel activity.

BACKGROUND & AIMS: Changes in mitochondrial energy metabolism promoted by uncoupling proteins (UCPs) are often found in metabolic disorders. We have recently shown that hypertriglyceridemic (HTG) mice present higher mitochondrial resting respiration unrelated to UCPs. Here, we disclose the underlying mechanism and consequences, in tissue and whole body metabolism, of this mitochondrial response to hyperlipidemia. METHODS: Oxidative metabolism and its response to mitochondrial adenosine triphosphate (ATP)-sensitive K+ channel (mitoK(ATP)) agonists and antagonists were measured in isolated mitochondria, livers, and mice. RESULTS: Mitochondria isolated from the livers of HTG mice presented enhanced respiratory rates compared with those from wild-type mice. Changes in oxygen consumption were sensitive to adenosine triphosphate (ATP), diazoxide, and 5-hydroxydecanoate, indicating they are attributable to mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) activity. Indeed, mitochondria from HTG mice presented enhanced swelling in the presence of K+ ions, sensitive to mitoK(ATP) agonists and antagonists. Furthermore, mitochondrial binding to fluorescent glibenclamide indicates that HTG mice expressed higher quantities of mitoK(ATP). The higher content and activity of liver mitoK(ATP) resulted in a faster metabolic state, as evidenced by increased liver oxygen consumption and higher body CO(2) release and temperature in these mice. In agreement with higher metabolic rates, food ingestion was significantly larger in HTG mice, without enhanced weight gain. CONCLUSIONS: These results show that primary hyperlipidemia leads to an elevation in liver mitoK(ATP) activity, which may represent a regulated adaptation to oxidize excess fatty acids in HTG mice. Furthermore, our data indicate that mitoK(ATP), in addition to UCPs, may be involved in the control of energy metabolism and body weight.

Animals↗
PubMed2006

Diazoxide protects against methylmalonate-induced neuronal toxicity.

Methylmalonic acidemia is an inherited metabolic disorder that leads to brain damage associated to the accumulation of methylmalonic acid (MMA) and impairment of energy metabolism. We demonstrate here that treatment with diazoxide, an agonist of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)), can prevent death promoted by treatment with MMA in PC12 cells and freshly prepared rat brain slices. This diazoxide effect was reversed by 5-hydroxydecanoate, a mitoK(ATP) antagonist, confirming it occurs due to the activity of this channel. Diazoxide was not capable of preventing inner membrane potential loss promoted by MMA and Ca(2+) in isolated mitochondria, indicating it does not directly prevent mitochondrial damage. Furthermore, diazoxide did not prevent respiratory inhibition in cells treated with MMA. Interestingly, we found that the mitochondrial inner membrane potential within intact cells treated with MMA was maintained in part by the reverse activity of ATP synthase (ATP hydrolysis) and that diazoxide prevented the formation of the membrane potential in the presence of MMA, in a manner sensitive to 5-hydroxydecanoate. Furthermore, the effects of diazoxide on cell survival after treatment with MMA were similar to those of ATP synthase inhibitor oligomycin and adenine nucleotide translocator inhibitor atractyloside. These results indicate that diazoxide prevents PC12 cell death promoted by MMA by decreasing mitochondrial ATP hydrolysis. These results uncover new potential neuroprotective effects of mitoK(ATP) agonists under situations in which oxidative phosphorylation is inhibited.

Adenosine Triphosphate↗
PubMed2005

Ischemic preconditioning requires increases in reactive oxygen release independent of mitochondrial K+ channel activity.

Mitochondrial ATP-sensitive K+ channels (mitoKATP) mediate ischemic preconditioning, a cardioprotective procedure. MitoKATP activity has been proposed to either enhance or prevent the release of reactive oxygen species. This study tested the redox effects of mitoKATP in order to clarify the role of these channels during preconditioning. We found no evidence that mitoKATP channels increase mitochondrial reactive oxygen species release directly. In addition, neither ischemic preconditioning nor the mitoKATP agonist diazoxide increased antioxidant defenses. Furthermore, increases in reactive oxygen species observed during ischemic preconditioning were not inhibited by mitoKATP antagonists, suggesting that they occur upstream of channel activity. Antioxidants were tested to verify if diazoxide-promoted ischemic protection was dependent on reactive oxygen species. N-Acetylcysteine proved to be an inadequate antioxidant for these tests since it directly interfered with the ability of diazoxide to activate mitoKATP. Catalase reversed the beneficial effect of preconditioning, but not of diazoxide, indicating that reactive oxygen species mediating preconditioning occur upstream of mitoKATP. Taken together, these results demonstrate that ischemic preconditioning increases reactive oxygen release independently of mitoKATP and suggest that the activity of this channel prevents oxidative reperfusion damage by decreasing reactive oxygen species production.

Acetylcysteine↗
PubMed2005

Tissue protection mediated by mitochondrial K+ channels.

Two distinct K+ uniporters have been described in mitochondria, ATP-sensitive and Ca2+-activated. Both are capable of protecting tissues against ischemia and other forms of injury when active. These findings indicate a central role for mitochondrial K+ uptake in tissue protection. This review describes the characteristics of mitochondrial K+ uniport, physiological consequences of this transport, forms of tissue damage in which K+ channels are implicated and possible mechanisms through which protection occurs.

Animals↗
PubMed2005

Mitochondrial ATP-sensitive K+ channels prevent oxidative stress, permeability transition and cell death.

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)) protects the heart against ischemic damage. This study examined the effects of mitoK(ATP) and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK(ATP) opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK(ATP) opening in isolated rat heart mitochondria slightly decreased Ca(2+) uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca(2+). In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK(ATP) prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca(2+) uptake, inhibiting mitochondrial permeability transition.

ATP-Binding Cassette Transporters↗
PubMed2004

Bcl-2 family proteins regulate mitochondrial reactive oxygen production and protect against oxidative stress.

Bcl-2 family proteins protect against a variety of forms of cell death, including acute oxidative stress. Previous studies have shown that overexpression of the antiapoptotic protein Bcl-2 increases cellular redox capacity. Here we report that cell lines transfected with Bcl-2 paradoxically exhibit increased rates of mitochondrial H(2)O(2) generation. Using isolated mitochondria, we determined that increased H(2)O(2) release results from the oxidation of reduced nicotinamide adenine dinucleotide-linked substrates. Antiapoptotic Bcl-2 family proteins Bcl-xL and Mcl-1 also increase mitochondrial H(2)O(2) release when overexpressed. Chronic exposure of cells to low levels of the mitochondrial uncoupler carbonyl cyanide 4-(triflouromethoxy)phenylhydrazone reduced the rate of H(2)O(2) production by Bcl-xL overexpressing cells, resulting in a decreased ability to remove exogenous H(2)O(2) and enhanced cell death under conditions of acute oxidative stress. Our results indicate that chronic and mild elevations in H(2)O(2) release from Bcl-2, Bcl-xL, and Mcl-1 overexpressing mitochondria lead to enhanced cellular antioxidant defense and protection against death caused by acute oxidative stress.

Animals↗
PubMed2004

Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae.

Increased replicative longevity in Saccharomyces cerevisiae because of calorie restriction has been linked to enhanced mitochondrial respiratory activity. Here we have further investigated how mitochondrial respiration affects yeast life span. We found that calorie restriction by growth in low glucose increased respiration but decreased mitochondrial reactive oxygen species production relative to oxygen consumption. Calorie restriction also enhanced chronological life span. The beneficial effects of calorie restriction on mitochondrial respiration, reactive oxygen species release, and replicative and chronological life span could be mimicked by uncoupling agents such as dinitrophenol. Conversely, chronological life span decreased in cells treated with antimycin (which strongly increases mitochondrial reactive oxygen species generation) or in yeast mutants null for mitochondrial superoxide dismutase (which removes superoxide radicals) and for RTG2 (which participates in retrograde feedback signaling between mitochondria and the nucleus). These results suggest that yeast aging is linked to changes in mitochondrial metabolism and oxidative stress and that mild mitochondrial uncoupling can increase both chronological and replicative life span.

Mitochondria↗
PubMed2004

Glutathione and thioredoxin peroxidases mediate susceptibility of yeast mitochondria to Ca(2+)-induced damage.

The effect of thioredoxin peroxidases on the protection of Ca(2+)-induced inner mitochondrial membrane permeabilization was studied in the yeast Saccharomyces cerevisiae using null mutants for these genes. Since deletion of a gene can promote several other effects besides the absence of the respective protein, characterizations of the redox state of the mutant strains were performed. Whole cellular extracts from all the mutants presented lower capacity to decompose H(2)O(2) and lower GSH/GSSG ratios, as expected for strains deficient for peroxide-removing enzymes. Interestingly, when glutathione contents in mitochondrial pools were analyzed, all mutants presented lower GSH/GSSG ratios than wild-type cells, with the exception of DeltacTPxI strain (cells in which cytosolic thioredoxin peroxidase I gene was disrupted) that presented higher GSH/GSSG ratio. Low GSH/GSSG ratios in mitochondria increased the susceptibility of yeast to damage induced by Ca(2+) as determined by membrane potential and oxygen consumption experiments. However, H(2)O(2) removal activity appears also to be important for mitochondria protection against permeabilization because exogenously added catalase strongly inhibited loss of mitochondrial potential. Moreover, exogenously added recombinant peroxiredoxins prevented inner mitochondrial membrane permeabilization. GSH/GSSG ratios decreased after Ca(2+) addition, suggesting that reactive oxygen species (ROS) probably mediate this process. Taken together our results indicate that both mitochondrial glutathione pools and peroxide-removing enzymes are key components for the protection of yeast mitochondria against Ca(2+)-induced damage.

Calcium↗
PubMed2004

A highly active ATP-insensitive K+ import pathway in plant mitochondria.

We describe here a regulated and highly active K+ uptake pathway in potato (Solanum tuberosum), tomato (Lycopersicon esculentum), and maize (Zea mays) mitochondria. K+ transport was not inhibited by ATP, NADH, or thiol reagents, which regulate ATP-sensitive K+ channels previously described in plant and mammalian mitochondria. However, K+ uptake was completely prevented by quinine, a broad spectrum K+ channel inhibitor. Increased K+ uptake in plants leads to mitochondrial swelling, respiratory stimulation, heat release, and the prevention of reactive oxygen species formation. This newly described ATP-insensitive K+ import pathway is potentially involved in metabolism regulation and prevention of oxidative stress.

Adenosine Triphosphate↗
PubMed2004

Phosphate increases mitochondrial reactive oxygen species release.

The effects of inorganic phosphate (Pi), the main intracellular membrane permeable anion capable of altering mitochondrial pH gradients (Delta pH), were measured on mitochondrial H2O2 release. As expected, Pi decreased Delta pH and increased the electric membrane potential (Delta Psi). Mitochondrial H2O2 release was stimulated by Pi and also by its structural analogue arsenate. However, acetate, another membrane-permeable anion, did not stimulate mitochondrial H2O2 release. The stimulatory effect promoted by Pi was prevented by CCCP, which decreases transport of Pi across the inner mitochondrial membrane, indicating that Pi must be in the mitochondrial matrix to stimulate H2O2 release. In conclusion, we found that Pi and arsenate stimulate mitochondrial reactive oxygen release, an effect that may contribute towards oxidative stress under conditions such as ischemia/reperfusion, in which high-energy phosphate bonds are hydrolyzed.

Animals↗
PubMed2004

Mitochondrial permeability transition in neuronal damage promoted by Ca2+ and respiratory chain complex II inhibition.

Changes in mitochondrial integrity, reactive oxygen species release and Ca2+ handling are proposed to be involved in the pathogenesis of many neurological disorders including methylmalonic acidaemia and Huntington's disease, which exhibit partial mitochondrial respiratory inhibition. In this report, we studied the mechanisms by which the respiratory chain complex II inhibitors malonate, methylmalonate and 3-nitropropionate affect rat brain mitochondrial function and neuronal survival. All three compounds, at concentrations which inhibit respiration by 50%, induced mitochondrial inner membrane permeabilization when in the presence of micromolar Ca2+ concentrations. ADP, cyclosporin A and catalase prevented or delayed this effect, indicating it is mediated by reactive oxygen species and mitochondrial permeability transition (PT). PT induced by malonate was also present in mitochondria isolated from liver and kidney, but required more significant respiratory inhibition. In brain, PT promoted by complex II inhibition was stimulated by increasing Ca2+ cycling and absent when mitochondria were pre-loaded with Ca2+ or when Ca2+ uptake was prevented. In addition to isolated mitochondria, we determined the effect of methylmalonate on cultured PC12 cells and freshly prepared rat brain slices. Methylmalonate promoted cell death in striatal slices and PC12 cells, in a manner attenuated by cyclosporin A and bongkrekate, and unrelated to impairment of energy metabolism. We propose that under conditions in which mitochondrial complex II is partially inhibited in the CNS, neuronal cell death involves the induction of PT.

Animals↗
PubMed2004

Binding, aggregation and photochemical properties of methylene blue in mitochondrial suspensions.

Methylene Blue (MB) has well-established photochemical properties and has been used in a variety of photochemical applications including photodynamic therapy. Despite the fact that most of MB's cytotoxic effects in cells are attributed to mitochondrial damage, the interactions of this dye with mitochondria and the consequent effects on photochemical properties have not yet been fully determined. We monitored MB binding, aggregation and its ability to release singlet oxygen (1O2) on irradiation when interacting with mitochondrial suspensions. MB actively binds to mitochondria and enters the matrix in a manner stimulated by the mitochondrial proton potential and by the increase in mitochondrial concentrations. The greater accumulation of MB in mitochondria with elevated proton potentials or those treated with high concentrations of MB results in the formation of MB dimers, previously shown to be less effective generators of 1O2. Accumulation of MB within mitochondria with high membrane potentials also results in the reduction of MB to the photochemically inactive leuco-MB. Indeed, irradiation of mitochondria with high proton potentials in the presence of MB results in the generation of approximately half the quantity of 1O2 compared with 1O2 generated in mitochondria with low proton potentials. These differences in photochemical properties should influence the cytotoxic effects of photodynamic treatment in the presence of MB.

Animals↗
PubMed2003

ATP-sensitive K+ channels in renal mitochondria.

Isolated kidney mitochondria swell when incubated in hyposmotic solutions containing K+ salts in a manner inhibited by ATP, ADP, 5-hydroxydecanoate, and glibenclamide and stimulated by GTP and diazoxide. These results suggest the existence of ATP-sensitive K+ channels in these mitochondria, similar to those previously described in heart, liver, and brain. Renal mitochondrial ATP-sensitive K+ uptake rates are approximately 140 nmol.min-1.mg protein-1. This K+ transport results in a slight increase in respiration and decrease in the inner membrane potential. In addition, the activation of ATP-inhibited K+ uptake using diazoxide leads to a decrease of ATP hydrolysis through the reverse activity of the F0F1 ATP synthase when respiration is inhibited. In conclusion, we characterize an ATP-sensitive K+ transport pathway in kidney mitochondria that affects volume, respiration, and membrane potential and may have a role in the prevention of mitochondrial ATP hydrolysis.

Adenosine Triphosphate↗