Adaphostin and other anticancer drugs quench the fluorescence of mitochondrial potential probes.

Fluorescent dyes are widely used to monitor changes in mitochondrial transmembrane potential (DeltaPsim). When MitoTracker Red CMXRos, tetramethylrhodamine methyl ester (TMRM), and 3,3'dihexyloxacarbocyanine iodide (DiOC6(3)) were utilized to examine the effects of the experimental anticancer drug adaphostin on intact cells or isolated mitochondria, decreased fluorescence was observed. In contrast, measurement of tetraphenylphosphonium uptake by the mitochondria using an ion-selective microelectrode failed to show any effect of adaphostin on DeltaPsim. Instead, further experiments demonstrated that adaphostin quenches the fluorescence of the mitochondrial dyes. Structure-activity analysis revealed that the adamantyl and p-aminobenzoic acid moieties of adaphostin are critical for this quenching. Anticancer drugs containing comparable structural motifs, including mitoxantrone, aminoflavone, and amsacrine, also quenched the mitochondrial probes. These results indicate the need for caution when mitochondrial dyes are utilized to examine the effects of xenobiotics on DeltaPsim and suggest that some previously reported direct effects of anticancer drugs on mitochondria might need re-evaluation.

Restoration of Ca2+-inhibited oxidative phosphorylation in cardiac mitochondria by mitochondrial Ca2+ unloading.

Mitochondria, the major source of cellular ATP, display high vulnerability to metabolic stress, in particular to excessive Ca2+ loading. Here, we show that Ca2+-inhibited mitochondrial ATP generation could be restored through stimulated Ca2+ discharge from mitochondrial matrix. This was demonstrated using a Ca2+ ionophore or through Na+/Ca2+ exchange-mediated decrease of mitochondrial Ca2+ load. Furthermore, diazoxide, a mitochondrial potassium channel opener, which maintained mitochondrial Ca2+ homeostasis, also restored Ca2+-inhibited ATP synthesis and preserved the structural integrity of Ca2+-challenged mitochondria. Thus, under conditions of excessive mitochondrial Ca2+ overload targeting mitochondrial Ca2+ transport pathways restores oxidative phosphorylation required for vital cellular processes. This study, therefore, identifies an effective strategy capable to rescue Ca2+-disrupted mitochondrial energetics.

Increased calcium vulnerability of senescent cardiac mitochondria: protective role for a mitochondrial potassium channel opener.

In senescence, endogenous mechanisms of cardioprotection are apparently attenuated resulting in increased vulnerability to ischemia-reperfusion. In particular, mitochondria, which are essential in maintaining cardiac energetic and ionic homeostasis, are susceptible to Ca2+ overload, a component of metabolic injury. However, effective means of protecting senescent mitochondria are lacking. Here, mitochondrial function and structure were assessed using ion-selective mini-electrodes, high-performance liquid chromatography and electron microscopy. Aging decreased ADP-induced oxygen consumption and prolonged the time associated with ADP to ATP conversion, which manifested as a reduced rate of oxidative phosphorylation. Aging also reduced mitochondrial Ca2+ handling, and increased Ca2+-induced mitochondrial damage. Diazoxide, a potassium channel opener, reduced Ca2+ loading and protected the functional and structural integrity of senescent mitochondria from Ca2+-induced injury. In this way, the present study identifies the potential usefulness for pharmacotherapy in protecting vulnerable senescent mitochondria from conditions of Ca2+ overload, such as ischemia-reperfusion.

Diazoxide protects mitochondria from anoxic injury: implications for myopreservation.

BACKGROUND: Heart muscle primarily relies on adenosine triphosphate produced by oxidative phosphorylation and is highly vulnerable to anoxic insult. Although a number of strategies aimed at improving myopreservation are available, no effective means of preserving mitochondrial energetics under conditions of anoxic injury have been developed. Openers of mitochondrial adenosine triphosphate-sensitive potassium channels have emerged as powerful cardioprotective agents presumably capable of maintaining mitochondrial function under metabolic stress. Here, we evaluated the ability of a prototype mitochondrial adenosine triphosphate-sensitive potassium channel opener, diazoxide, to preserve oxidative phosphorylation in mitochondria subjected to anoxia and reoxygenation. METHODS: Mitochondria were isolated from rat hearts and subjected to 20 minutes of anoxia, followed by reoxygenation. Mitochondrial respiration and oxidative phosphorylation, as well as mitochondrial integrity, were assessed by means of ion-selective minielectrodes, high-performance liquid chromatography, fluorometry, and electron microscopy. RESULTS: Anoxia-reoxygenation decreased the rate of adenosine diphosphate-stimulated oxygen consumption, inhibited adenosine triphosphate production, and disrupted mitochondrial integrity. On average, anoxic stress reduced adenosine diphosphate-stimulated respiration from 291 +/- 14 to 141 +/- 15 ng-atoms O(2). min(-1). mg(-1) protein and decreased the rate of adenosine triphosphate production from 752 +/- 14 to 414 +/- 34 nmol adenosine triphosphate. min(-1). mg(-1) protein. After anoxia, the majority (88%) of mitochondria was damaged or swollen and released adenylate kinase, a marker of mitochondrial integrity. Diazoxide (100 micromol/L), present throughout anoxia, preserved adenosine diphosphate-stimulated respiration at 255 +/- 7 ng-atoms O(2). min(-1). mg(-1) protein and adenosine triphosphate production at 640 +/- 39 nmol adenosine triphosphate. min(-1). mg(-1) protein. Diazoxide also protected mitochondrial structure from anoxia-mediated damage, so that after anoxic stress, 67% of mitochondria remained intact and adenylate kinase was confined to the mitochondria. CONCLUSIONS: The present study demonstrates that diazoxide diminishes anoxia-induced functional and structural deterioration of cardiac mitochondria. By protecting mitochondria and preserving myocardial energetics, diazoxide may be useful under conditions of reduced oxygen availability, including global surgical ischemia or storage of donor heart.

ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria.

1. Mitochondrial dysfunction, secondary to excessive accumulation of Ca2+, has been implicated in cardiac injury. We here examined the action of potassium channel openers on mitochondrial Ca2+ homeostasis, as these cardioprotective ion channel modulators have recently been shown to target a mitochondrial ATP-sensitive K+ channel. 2. In isolated cardiac mitochondria, diazoxide and pinacidil decreased the rate and magnitude of Ca2+ uptake into the mitochondrial matrix with an IC50 of 65 and 128 microM, respectively. At all stages of Ca2+ uptake, the potassium channel openers depolarized the mitochondrial membrane thereby reducing Ca2+ influx through the potential-dependent mitochondrial uniporter. 3. Diazoxide and pinacidil, in a concentration-dependent manner, also activated release of Ca2+ from mitochondria. This was prevented by cyclosporin A, an inhibitor of Ca2+ release through the mitochondrial permeability transition pore. 4. Replacement of extramitochondrial K+ with mannitol abolished the effects of diazoxide and pinacidil on mitochondrial Ca2+, while the K+ ionophore valinomycin mimicked the effects of the potassium channel openers. 5. ATP and ADP, which block K+ flux through mitochondrial ATP-sensitive K+ channels, inhibited the effects of potassium channel openers, without preventing the action of valinomycin. 6. In intact cardiomyocytes, diazoxide also induced mitochondrial depolarization and decreased mitochondrial Ca2+ content. These effects were inhibited by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. 7. Thus, potassium channel openers prevent mitochondrial Ca2+ overload by reducing the driving force for Ca2+ uptake and by activating cyclosporin-sensitive Ca2+ release. In this regard, modulators of an ATP-sensitive mitochondrial K+ conductance may contribute to the maintenance of mitochondrial Ca2+ homeostasis.

Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function.

Discovered in the cardiac sarcolemma, ATP-sensitive K+ (KATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial KATP channel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K+ channel openers to examine the effect of mitochondrial KATP channel opening on mitochondrial membrane potential, respiration, ATP generation, Ca2+ transport, and matrix volume. From a mitochondrial membrane potential of -180 +/- 15 mV, K+ channel openers, pinacidil (100 microM), cromakalim (25 microM), and levcromakalim (20 microM), induced membrane depolarization by 10 +/- 7, 25 +/- 9, and 24 +/- 10 mV, respectively. This effect was abolished by removal of extramitochondrial K+ or application of a KATP channel blocker. K+ channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K+ channel opener released Ca2+ from mitochondria preloaded with Ca2+, an effect also dependent on extramitochondrial K+ concentration and sensitive to KATP channel blockade. In addition, K+ channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome c and adenylate kinase. Thus, in isolated cardiac mitochondria, KATP channel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca2+, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial KATP channels in regulating functions vital for the cardiac mitochondria.

A model of mitochondrial Ca(2+)-induced Ca2+ release simulating the Ca2+ oscillations and spikes generated by mitochondria.

Recent evidence underlines a key role of mitochondrial Ca2+ fluxes in cell Ca2+ signalling. We present here a kinetic model simulating the Ca2+ fluxes generated by mitochondria during mitochondrial Ca(2+)-induced Ca2+ release (mCICR) resulting from the operation of the permeability transition pore (PTP). Our model connects the Ca2+ fluxes through the ruthenium redsensitive Ca2+ uniporter, the respiration-dependent and passive H+ fluxes, the rate of oxygen consumption, the movements of weak acids across the mitochondrial membrane, the electrical transmembrane potential (delta psi), and operation of the PTP. We find that two factors are crucial to account for the various mCICR profiles that can be observed experimentally: (i) the dependence of PTP opening and closure on matrix pH (pHi), and (ii) the relative inhibition of the respiratory rate consecutive to PTP opening. The resulting model can simulate irreversible Ca2+ efflux from mitochondria, as well as the genesis of damped or sustained Ca2+ oscillations, and of single Ca2+ spikes. The model also simulates the main features of mCICR, i.e. the threshold-dependence of mCICR triggering, and the all-or-nothing nature of mCICR operation. Our model should appear useful to further mathematically address the consequences of mCICR on the spatiotemporal organisation of Ca2+ signals, as a 'plug-in' module for the existing models of cell Ca2+ signalling.

Non-cholinergic toxicity of organophosphates in mammals: interaction of ethaphos with mitochondrial functions.

The effect of the organophosphorus insecticide O-ethyl-S-propyl-2,4-dichlorophenylphosphorothioate (ethaphos) on mitochondrial functions was studied. It was shown that ethaphos interacts with mitchondrial oxidative metabolism and inhibits oxidation of succinate, alpha-glycerophosphate and pyruvate+malate at concentrations of 100, 75 and 50 micrograms mg-1 mitochondrial protein, respectively. Mitochondria treated with ethaphos show no changes in the inner membrane permeability and activity of cytochromes of the respiratory chain. Ethaphos has no inhibitory effect on the activity of mitochondrial ATPase. Addition of the electron donor pair (ascorbic acid + N,N,N',N'-tetramethyl-p-phenylenediamine) to ethaphostreated mitochondria restored the respiration and membrane potential. The membrane potential also can be re-established in poisoned mitochondria by the addition of exogenous ATP. Based on the data obtained we show that mitochondrial dysfunctions induced by ethaphos can be partially eliminated and mitochondrial functions can be restored using artificial electron donors and/or through increasing the cytosolic ATP level.

Synchronization of calcium waves by mitochondrial substrates in Xenopus laevis oocytes.

In Xenopus oocytes, as well as other cells, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release is an excitable process that generates propagating Ca2+ waves that annihilate upon collision. The fundamental property responsible for excitability appears to be the Ca2+ dependency of the Ins(1,4,5)P3 receptor. Here we report that Ins(1,4,5)P3-induced Ca2+ wave activity is strengthened by oxidizable substrates that energize mitochondria, increasing Ca2+ wave amplitude, velocity and interwave period. The effects of pyruvate/malate are blocked by ruthenium red at the Ca2+ uniporter, by rotenone at complex I, and by antimycin A at complex III, and are subsequently rescued at complex IV by ascorbate tetramethylphenylenediamine (TMPD). Our data reveal that potential-driven mitochondrial Ca2+ uptake is a major factor in the regulation of Ins(1,4,5)P3-induced Ca2+ release and clearly demonstrate a physiological role of mitochondria in intracellular Ca2+ signalling.

Strontium excitability of the inner mitochondrial membrane: regenerative strontium-induced strontium release.

Regenerative Sr(++)-induced Sr++ release from isolated rat liver mitochondria was studied using ion-selective electrode techniques. Mitochondria, when exposed to a pulse of Sr++, demonstrated a reversible and transient increase in inner membrane permeability to K+ and H+ ions. The increase in permeability was an all-or-none process with a threshold dependent on the amplitude of the Sr++ pulse. The threshold concentration of Sr++ was lowered from 120-150 microM to 20-30 microM when mitochondria were preloaded with 100 nmoles Sr++/mg protein. Release of matrix-stored divalent cations provided a mechanism for amplification of the extramitochondrial Sr++ concentration (regenerative Sr(++)-induced Sr++ release). The mitochondrial inner membrane became refractory, since subsequent cycles of excitation could not be immediately induced. These experiments demonstrate that the inner mitochondrial membrane is an excitable membrane, with threshold-dependent, regenerative and subsequently refractile Sr(++)-induced Sr++ release characteristics.

Nucleotides-induced cytosolic calcium transient and plasma membrane permeability in Chang human liver cells.

Extracellular nucleotides induce changes in cytosolic free Ca++ and also increase plasma membrane permeability to Ca++ ions in Chang human liver cells. Ca++ permeability induced by nucleotides is reversible and inactivated immediately upon removal of agonist. Stimulated cells transferred into the fresh medium and re-exposed to nucleotides demonstrate reopening of Ca++ channel without stimulation of Ca++ transient. Relative potencies of nucleotides to induce membrane permeability and Ca++ transient were: UTP > ATP > gamma-S-ATP > ADP (non-hydrolyzable ATP analogues and AMP had no effect). The permeability is not affected by specific inhibitors of voltage-operated calcium channels (verapamyl,cis-diltiazem and nifedipin). Nucleotides do not produce plasma membrane damages at concentrations up to 1 mM, as shown by exclusion of the propidium iodide. There are at least two types of nucleotides receptors in Chang cell membrane: P2y subtype receptors which is responsible for generation of the Ca++ transient, and P2x subtype receptors which lead to the opening of plasma membrane Ca++ channels upon activation.

Mitochondrial calcium spiking: a transduction mechanism based on calcium-induced permeability transition involved in cell calcium signalling.

We report reversible Ca(2+)-induced Ca2+ release from mitochondria, which takes the form of Ca2+ spikes. Mitochondrial Ca2+ spiking is an all-or-none process with a threshold dependence on both the frequency and the amplitude of the Ca2+ pulses used as stimuli. This spiking relies on the transient operation of the mitochondrial permeability transition pore, and is initiated--in a threshold-dependent manner--with inositol-triphosphate-mediated Ca2+ responses on permeabilized cells. Evidence that mitochondrial Ca(2+)-induced Ca2+ release contributes to inositol-triphosphate-mediated Ca2+ responses in intact cells is also reported.

Oscillation of ion fluxes in mammalian erythrocytes. Mechanism of oscillation.

The dependence of ionophore-induced oscillations in rat erythrocytes on various concentrations of A23187, FCCP and Ca2+ was analysed using ion-selective electrodes. The oscillations were shown to be independent of the extracellular concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone and Ca2+. The dependence of oscillations on the concentration A23187 was shown to be a threshold characteristic and represented by a bell-shaped curve. In the course of oscillations the redistribution of A23187 between cells and the incubation medium was demonstrated using high-speed centrifugation. A hypothesis for oscillatory-state generation in erythrocytes was suggested on the basis of pH-dependent changes of the Ca2+ ionophore A23187 content in cells. According to this hypothesis the H+ concentration within the external membrane-adjacent layer serves as a causative factor for induction of cyclic desorption of A23187 molecules from the cell membrane.

Oscillating dissipative structures in mitochondrial suspensions.

The occurrence of spatial structures in the unstirred layer of an oscillating mitochondrial suspension is reported. The structures are detected by photo camera by light scattering in the unstirred layer of suspension. The spatial structures observed are shown to oscillate with the same period as that of mitochondrial oscillations in the bulk phase. Patterning is not affected by the layer depth within the range 0.3-3.0 mm. Various types of oscillatory states of mitochondria are characterized by the corresponding patterns. Patterning is effectively suppressed by the inhibitors of the respiratory chain (antimycin A or CN-).

Spatial structures in mitochondrial suspension induced by cation efflux.

The formation of spatial structures in a thin unstirred layer of a mitochondrial suspension has been studied. It is shown that the structure formation depends on the state of the ion-transporting systems of mitochondria and that pattern development coincides with the activation of cation efflux from preloaded mitochondria. Spatial structure formation is an energy-dependent process and is suppressed by respiratory chain inhibitors. Patterning is also inhibited by EGTA, EDTA and ruthenium red, reflecting the requirement for divalent cation translocation in mitochondria for the studied phenomenon.

The influence of starvation on some characteristics of the Ca2+ transport system and lipid content in rat liver mitochondria.

The effects of 48-hour starvation on some characteristics of the Ca2+ transport system as well as on lipid content and free fatty acids composition in rat liver mitochondria were determined. The ion fluxes in mitochondria in steady state and oscillations were measured using Ca2+, Sr2+ and H+ sensitive electrodes. The Ca2+ uptake in liver mitochondria was changed after starvation. In the case of equal amounts of endogenous mitochondrial Ca2+ the capability of liver mitochondria to accumulate and store exogenous Ca2+ was decreased after starvation. After inhibition of the energy dependent (active) Ca2+ transport by ruthenium-red (RR) the rate of the passive Ca2+ efflux was activated and this could be explained by the induction of the electroneutral 2H+/Me2+ exchange after starvation. The disproportion in the amounts of linoleic and docosahexaenoic acids in mitochondrial phospholipids after starvation is considered to be the possible cause of the changes in the structure and permeability of the mitochondrial membrane.

Effect of pH and proton buffer on oscillations of ion fluxes in rat erythrocytes.

The pH-dependence of ionophore-induced oscillations of transmembrane H+ and K+ fluxes in rat erythrocytes has been studied. It is stated that the oscillations are strongly depressed at pH lower than 6.9 and higher than 7.3. Proton buffers of different nature (Tris/HCl, Mops and glycylglycine) are shown to effectively inhibit the oscillatory process. The significance of H+ concentration in unstirred layers adjacent to the membrane in the mechanism of oscillations is suggested.

Induction of 2H+/Me2+ exchange in rat-liver mitochondria.

The time dependency of CA2+ efflux from Ca2+-loaded rat liver mitochondria has been investigated. The rate of ruthenium-red-insensitive Ca2+ efflux is continuously increased during the retention as a result of induction of an electroneutral H+ Ca2+ exchange system. The activation of the Ca2+ efflux pathway takes place under the constant value of the membrane potential and is accompanied by oxidation of mitochondrial pyridine nucleotides. It has also been found that the ruthenium-red-insensitive H+/Sr2+ exchange occurs in mitochondria during Sr2+-induced oscillation of ion fluxes. The rate of H+/Sr2+ exchange is variable and depends on the stage of the oscillatory cycle.