Effects of doxorubicin and its aglycone metabolite on calcium sequestration by rabbit heart, liver, and kidney mitochondria

Previous investigators have shown that following doxorubicin treatment heart mitochondria appear swollen and contain intramitochondrial dense inclusion bodies identified as calcium phosphate. In vitro studies have shown that similar morphological changes occur in mitochondria previously loaded with excess calcium. The present studies were performed to determine the effects of doxorubicin and its aglycone metabolite on ⁴⁵Ca²⁺ uptake by mitochondria isolated from the heart, liver, and kidney of the rabbit. Doxorubicin (100 μM) significantly inhibited the initial rate of ⁴⁵Ca²⁺ accumulated by mitochondria isolated from the three tissues. In contrast, the aglycone metabolite (100 μM) induced the reverse effect. In preloaded mitochondria the aglycone stimulated the release of calcium while doxorubicin was without effect. Mitochondria from the heart were significantly more sensitive to the effects of these anthracyclines than were mitochondria from the other two tissues. If these in vitro effects also occur in vitro, then the aglycone metabolite would be a more likely candidate in explaining the morphological changes in heart mitochondria previously described.     

Oligosaccharides of human milk. Isolation and characterization of three new disialyfucosyl hexasaccharides.

Renal mitochondrial 25-hydroxyvitamin D₃-1-hydroxylase (1-hydroxylase) is sensitive to inhibition by 2 × 10^?5m calcium and 5 × 10^?3m phosphate when hydroxylation is supported by either malate or NADPH. This sensitivity to ion inhibition is observed in mitochondria from both vitamin D-deficient and repleted chicks and remains when mitochondria are frozen and thawed or are incubated in a hypotonic medium. The ionophore A23187 inhibits the 1-hydroxylase but partially reverses the inhibition exerted by 2, 5, or 7.5 × 10^?5m calcium. Addition of a kidney soluble cell fraction (37,000g supernatant) to isolated mitochondria did not enhance the 1-hydroxylase activity under conditions of varied substrate concentration, osmolarity of the incubation medium, or mitochondrial washes. It is concluded that a soluble cellular component is not involved in the regulation of the 1-hydroxylase but that intramitochondrial calcium and phosphate may well play a role in its regulation.     

The Phosphate Source Influences Gene Expression and Quality of Mineralization during In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells

For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of β-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost- and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design.     

The effects of chlorotetracycline on calcium movements in isolated rat liver mitochondria

Chlorotetracycline was used as a fluorescent chelate probe for visualizing calcium movements in rat liver mitochondria. It was demonstrated that under specified conditions, chlorotetracycline-associated fluorescence may be employed as a monitor of calcium uptake by mitochondrial membranes, e.g., at low calcium and Chlorotetracycline concentrations and in the absence of exogenous phosphate or acetate. However, at elevated calcium concentrations, e.g., >0.05 mm, a transient fluorescence response was observed upon addition of calcium to energized mitochondria. This transient or cyclic behavior of the chlorotetracycline-associated fluorescence was minimized by increasing the chlorotetracycline concentration, the mitochondrial protein concentration, or by including magnesium in the incubation. Also, it was demonstrated that chlorotetracycline addition to mitochondria which had been loaded previously with ⁴⁵Ca resulted in a rapid efflux of the accumulated ⁴⁵Ca. Because of the various effects of chlorotetracycline on the ability of the mitochondria to accumulate and to retain calcium, caution must be exercised in the interpretation of experimental results when this fluorescent chelate probe is utilized to monitor the association of divalent metal cations with biological membranes.     

Evidence for two compartments of exchangeable calcium in isolated rat liver mitochondria obtained using a45Ca exchange technique in the presence of magnesium,phosphate, and ATP at 37°C

Summary The distribution of calcium between isolated rat liver mitochondria and the extramitochondrial medium at 37°C and in the presence of 2mm inorganic phosphate, 3mm ATP, 0.05 or 1.1mm free magnesium and a calcium buffer, nitrilotriacetic acid, was investigated using a⁴⁵Ca exchange technique. The amounts of⁴⁰Ca in the mitochondria and medium were allowed to reach equilibrium before initiation of the measurement of⁴⁵Ca exchange. At 0.05mm free magnesium and initial extramitochondrial free calcium concentrations of between 0.15 and 0.5 m, the mitochondria accumulated calcium until the extramitochondrial free calcium concentration was reduced to 0.15 m. Control experiments showed that the mitochondria were stable under the incubation conditions employed. The⁴⁵Ca exchange data were found to be consistent with a system in which two compartments of exchangeable calcium are associated with the mitochondria. Changes in the concentration of inorganic phosphate did not significantly affect the⁴⁵Ca exchange curves, whereas an increase in the concentration of free magnesium inhibited exchange. The maximum rate of calcium outflow from the mitochondria was estimated to be 1.7 nmol/min per mg of protein, and the value ofK _0.5 for intramitochondrial exchangeable calcium to be about 1.6 nmol per mg of protein. Ruthenium Red decreased the fractional transfer rate for calcium inflow to the mitochondria while nupercaine affected principally the fractional transfer rates for the transfer of calcium between the two mitochondrial compartments. The use of the incubation conditions and⁴⁵Ca exchange technique described in this report for studies of the effects of agents which may alter mitochondrial calcium uptake or release (e.g., the pre-treatment of cells with hormones) is briefly discussed.     

Dimeric H-ATP synthase in the chloroplast of Chlamydomonas reinhardtii

H⁺-ATP synthase is the dominant ATP production site in mitochondria and chloroplasts. So far, dimerization of ATP synthase has been observed only in mitochondria by biochemical and electron microscopic investigations. Although the physiological relevance remains still enigmatic, dimerization was proposed to be a unique feature of the mitochondrion [Biochim. Biophys. Acta 1555 (2002) 154]. It is hard to imagine, however, that closely related protein complexes of mitochondria and chloroplast should show such severe differences in structural organization. We present the first evidences for dimerization of chloroplast ATP synthases within the thylakoid membrane.By investigation of the thylakoid membrane of Chlamydomonas reinhardtii by blue-native polyacrylamide gel electrophoresis, dimerization of the chloroplast ATP synthase was detected. Chloroplast ATP synthase dimer dissociates into monomers upon incubation with vanadate or phosphate but not by incubation with molybdate, while the mitochondrial dimer is not affected by the incubation. This suggests a distinct dimerization mechanism for mitochondrial and chloroplast ATP synthase. Since vanadate and phosphate bind to the active sites, contact sites located on the hydrophilic CF₁ part are suggested for the chloroplast ATP synthase dimer. As the degree of dimerization varies with phosphate concentration, dimerization might be a response to low phosphate concentrations.     

Critical Parameters of the In Vitro Method of Vascular Smooth Muscle Cell Calcification

Background

Vascular calcification (VC) is primarily studied using cultures of vascular smooth muscle cells. However, the use of very different protocols and extreme conditions can provide findings unrelated to VC. In this work we aimed to determine the critical experimental parameters that affect calcification in vitro and to determine the relevance to calcification in vivo.

Experimental Procedures and Results

Rat VSMC calcification in vitro was studied using different concentrations of fetal calf serum, calcium, and phosphate, in different types of culture media, and using various volumes and rates of change. The bicarbonate content of the media critically affected pH and resulted in supersaturation, depending on the concentration of Ca²⁺ and Pi. Such supersaturation is a consequence of the high dependence of bicarbonate buffers on CO₂ vapor pressure and bicarbonate concentration at pHs above 7.40. Such buffer systems cause considerable pH variations as a result of minor experimental changes. The variations are more critical for DMEM and are negligible when the bicarbonate concentration is reduced to ¼. Particle nucleation and growth were observed by dynamic light scattering and electron microscopy. Using 2mM Pi, particles of ~200nm were observed at 24 hours in MEM and at 1 hour in DMEM. These nuclei grew over time, were deposited in the cells, and caused osteogene expression or cell death, depending on the precipitation rate. TEM observations showed that the initial precipitate was amorphous calcium phosphate (ACP), which converts into hydroxyapatite over time. In blood, the scenario is different, because supersaturation is avoided by a tightly controlled pH of 7.4, which prevents the formation of PO₄ ^3--containing ACP.

Conclusions

The precipitation of ACP in vitro is unrelated to VC in vivo. The model needs to be refined through controlled pH and the use of additional procalcifying agents other than Pi in order to reproduce calcium phosphate deposition in vivo.     

The effect of inorganic phosphate on creatine kinase in respiring rat heart mitochondria

The ability of creatine to stimulate the respiration of rat heart mitochondria in vitro is reversibly affected by the concentration of inorganic phosphate. The rate of oxygen consumption due to post-ADP state-4 respiration in the presence of 20 mm creatine is reduced significantly when the potassium phosphate concentration is raised from 5 to 20 mm. State-3 respiration is reduced only by potassium phosphate concentrations higher than 20 mm. The rate of synthesis of creatine phosphate is also affected by phosphate concentration, and the apparent K_m of the coupled reactions for ADP is significantly higher at 25 mm phosphate as compared to that at 5 mm phosphate. These observations are consistent with the hypothesis that inorganic phosphate acts as an effector molecule, regulating creatine phosphate synthesis by favoring the dissociation of mitochondrial creatine kinase from the mitochondrial membrane. Such regulation may be important in the case of cells undergoing partial or severe ischemia, where changes in phosphate concentration within this range have been reported.     

Effects of physiological manipulation on the kinetics of mitochondrial phosphate transport in Saccharomyces cerevisiae

The kinetics of [³²P]phosphate uptake has been studied in different types of Saccharomyces cerevisiae mitochondria. Mitochondria were isolated from yeast grown aerobically on 2% lactate (Lac-mitochondria), 2% galactose (Gal-mitochondria), 5.4% glucose (Glu-mitochondria) or from yeast grown anaerobically on 2% galactose (Promitochondria). The effect of chloramphenicol was also studied by adding it to the growth medium of yeast grown aerobically on 2% galactose (chloramphenicol-mitochondria).[³²P]Phosphate uptake followed an oscillatory pattern in Lac, Gal-mitochondria and Promitochondria.Saturation kinetics were detected in fully differenciated mitochondria and in Promitochondria, but not in chloramphenicol-mitochondria.Glu-mitochondria did not translocate phosphate as shown both by lack of [³²P]phosphate uptake and lack of swelling in isoosmotic potassium solution.Repressed yeast cells were incubated in a resting cell medium and mitochondria were isolated at different times of incubation. The rate of respiration and the oligomycin-sensitive ATPase increased during the course of the incubation. After 2h, a mitochondrial mersalyl-sensitive swelling in an isoosmotic potassium phosphate solution was detected.As expected, no increase of the rate of respiration was observed when chloramphenicol was added in the derepression medium. But the oligomycin-sensitive ATPase decreased. Chloramphenicol did not affect the phosphate transport activity as measured by the swelling of mitochondria, but the [³²P]phosphate uptake did not follow saturation kinetics. A complete derepression of the inorganic phosphate-carrier activity was achieved by a 4 h incubation of the repressed cells in the presence of chloramphenicol, followed by a 6 h incubation in presence of cycloheximide.These data strongly suggest that the mitochondrial protein-synthesis system is required for the normal function of the inorganic phosphate-carrier.     

The calcium-binding glycoprotein and mitochondrial calcium movements

The calcium-binding glycoprotein isolated from mitochondria can be shown to move from one mitochondrial compartment to another as a function of calcium and magnesium presence as well as calcium transport. The movement is reversible $\text{in}\text{vitro}$ and the possibility is therefore considered that the glycoprotein may behave as a mobile calcium-carrier. In the presence of acetate and phosphate, calcium-pre-loaded mitochondria release the cation upon addition of uncoupling concentrations of pentachlorophenol. The rate of calcium efflux can be modulated either by changing pentachlorophenol or phosphate concentrations. Simultaneously a release of calcium-binding glycoprotein can be detected and a negative linear relation has been found between amount of glycoprotein released and rate of calcium passive efflux. The data are interpreted to indicate that calcium efflux occurs only when the glycoprotein is bound to the mitochondrial membranes.     

Varying effects of calcium on the oxidation of palmitate and α-ketoglutarate in isolated rat liver mitochondria incubated in KCl-based and sucrose-based media

• 1.1. Isolated mitochondria from rat liver were incubated in the presence of [U-¹⁴C]palmitate, ATP, CoA, carnitine, EGTA (ethylene glycol bis (β-aminoethyl ether) N,N′-tetraacetic acid) and varying amounts of calcium.
• 2.2. When a KCl-based incubation medium was used, the oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1–10μM.
• 3.3. When a sucrose-based incubation medium was used, the basal rate of palmitate oxidation was about half of that observed with the KCl-medium and calcium had a stimulatory effect.
• 4.4. With the KCl-medium the rate of oxygen consumption was inhibited by calcium with α-ketoglutarate as well as palmitate as the respiratory substrate.
• 5.5. No inhibitory effect of calcium was observed with succinate or β-hydroxybutyrate.
• 6.6. With the KCl-medium and with α-ketoglutarate as the respiratory substrate, state 3 respiration but not state 4 respiration was inhibited by calcium.
• 7.7. When the sucrose-medium was used, state 3 respiration was first inhibited by calcium, but this inhibition was gradually relieved and the respiratory rate finally became higher than it was before calcium addition.

     

Uptake of calcium in chromaffin granules of bovine adrenal medulla stimulated in vitro

The calcium content of bovine adrenal medulla perfused in vitro has been shown to increase about 30% in response to extensive acetylcholine stimulation. The calcium accumulated during secretion was mainly associated with the mitochondria and chromaffin granule fractions and to a lesser extent in the microsome fraction. While the calcium taken up by the mitochondria and microsomes was partly or totally removed by treatment with EDTA, the chelating agent had no effect on the granule content of calcium. The uptake of calcium in the mitochondria and microsomes during secretion is consistent with a function of these organelles in regulating the cellular calcium concentration. It is suggested that also the chromaffin granules may act as a “Ca-pump” in the chromaffin cell of the adrenal medulla.     

Surface reactivity and in vitro biological evaluation of sol gel derived silver/calcium silicophosphate bioactive glass

Ag ions are known for their antibacterial effects. Ag containing silicate glasses have been extended to create bioactive glasses that exhibit inhibitory effects on bacterial growth using different techniques. In this work, calcium and calcium/silver silicophosphate glasses were synthesized from the sol-gel process and their physicochemical and in vitro biological properties were studied and compared. The effect of silver concentration on in vitro bioactivity and antibacterial properties of the glasses was investigated. Ag₂O was substituted for CaO in the glass formula up to 2 mol% and in vitro bioactivity of the samples was evaluated by soaking them in simulated body fluid followed by structural characterization using XRD, FTIR and SEM techniques. The results showed that both glasses favored precipitation of the calcium phosphate layer when they were soaked in simulated body fluid; however, the morphology of apatite crystals changed for the 2% mol silver containing sample. Substitution of 2% mol Ag₂O for CaO seemed to slightly stimulate the rate of precipitation. The in vitro biodegradation rate of the silver/ calcium silicophosphate glasses was lower than that of the silver-free one (control). Also, the antibacterial properties of the samples indicated that these effects were improved by increasing silver concentration in bioactive glass composition.     

Oxidation of reduced nicotinamide adenine dinucleotide phosphate by isolated corn mitochondria

Isolated corn (Zea mays L.) mitochondria were found to oxidize reduced nicotinamide adenine dinucleotide phosphate in a KCl reaction medium. This oxidation was dependent on the presence of calcium or phosphate or both. Strontium and manganese substituted for calcium, but magnesium or barium did not. The oxidation of NADPH produced contraction of mitochondria swollen in KCl. Further evidence that the oxidation of NADPH was coupled was observed in respiratory control and adenosine diphosphate-oxygen ratios that were comparable to those reported for reduced nicotinamide adenine dinucleotide. The pathways of electron flow from NADH and NADPH were compared through the addition of electron transport inhibitors. The only difference between the two dinucleotides was that amytal was found to inhibit almost totally the state 3 oxidation of NADPH, but had little effect on the state 3 oxidation of NADH. The hypothetical pathways for electron flow from NADPH are discussed, as are the possible sites of calcium and phosphate stimulation.     

The effects of hydrogen ions on the kinetics of calcium transport by rat kidney mitochondria

The influence of pH on the kinetics of the initial rate of calcium uptake by isolated kidney mitochondria was studied using the ruthenium red-ethylene glycol bis(β-aminoethyl ether) N,N-tetraacetic acid quench method (K. Reed and F. Bygrave, 1975, Eur. J. Biochem.55, 497–504). In the absence of phosphate, the K_m is increased 50% and the V is decreased 57% when the pH is decreased from pH 7.4 to 7.0. Conversely, when the pH is increased to 7.8, the K_m is decreased 25% while the V is not affected. The presence of 0.1 or 0.4 mm phosphate in the incubation medium abolishes the change in K_m at a low pH while the V remains depressed by 36 and 25%, respectively. The presence of phosphate does not affect the decrease in the K_m seen with an increased medium pH. Mitochondria incubated in steady-state conditions with a medium free calcium of 0.7 μm also show significant changes in calcium exchange and distribution with pH. Two kinetic calcium pools are found in isolated mitochondria. Decreasing pH from 7.4 to 7.1 decreases mitochondria total calcium 32%, decreases the rapidly exchanging pool 28%, and depresses both the mitochondrial membrane and an intramitochondrial calcium exchange by 54 and 22%, respectively. Raising the pH to 7.7 increases both exchangeable pools (63 and 46%), and increases the mitochondrial membrane calcium exchange 44%. These results are consistent with previous studies on the influence of intracellular pH on calcium metabolism of kidney cells in which the mitochondrial pool was markedly affected by pH (R. Studer, and A. Borle, 1979, J. Membrane Biol.48, 325–341). Alterations in cellular pH may modify mitochondrial calcium transport and cellular calcium metabolism and thus affect cell functions which are calcium dependent.     

Calcium-activated phosphate uptake in contracting corn mitochondria

The phosphate inhibition of succinate-powered contraction in corn mitochondria can be reversed with calcium. Associated with this reversal is an accumulation of phosphate and calcium. Both ions are essential for accumulation, although strontium will partially substitute for calcium. Arsenate does not substitute for phosphate except in producing the inhibition of contraction.

The antibiotics oligomycin and aurovertin do not block the phosphate inhibition of contraction or the calcium-activated phosphate uptake associated with the release of the inhibition. Dinitrophenol uncouples the phosphate uptake but permits full contraction.

Calcium promotes inorganic phosphate accumulation in root tissue as well as in mitochondria.

The results are discussed from the viewpoint of theories of calcium reaction with high energy intermediates of oxidative phosphorylation. It is concluded that calcium probably reacts with X~P in corn mitochondria, rather than with X~I as with animal mitochondria.

     

The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions

Background

Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated.

Methods

Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS.

Results

It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents.

Conclusion

These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization.

General significance

This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.     

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis¹. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol². It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology³. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)⁴ or aequorin⁵ targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of ''ratiometric-pericam'' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin⁴. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 μM⁴. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.     

Transient Mitochondrial Depolarizations Reflect Focal Sarcoplasmic Reticular Calcium Release in Single Rat Cardiomyocytes

Digital imaging of mitochondrial potential in single rat cardiomyocytes revealed transient depolarizations of mitochondria discretely localized within the cell, a phenomenon that we shall call “flicker.” These events were usually highly localized and could be restricted to single mitochondria, but they could also be more widely distributed within the cell. Contractile waves, either spontaneous or in response to depolarization with 50 mM K⁺, were associated with propagating waves of mitochondrial depolarization, suggesting that propagating calcium waves are associated with mitochondrial calcium uptake and consequent depolarization. Here we demonstrate that the mitochondrial flicker was directly related to the focal release of calcium from sarcoplasmic reticular (SR) calcium stores and consequent uptake of calcium by local mitochondria. Thus, the events were dramatically reduced by (a) depletion of SR calcium stores after long-term incubation in EGTA or thapsigargin (500 nM); (b) buffering intracellular calcium using BAPTA-AM loading; (c) blockade of SR calcium release with ryanodine (30 μM); and (d) blockade of mitochondrial calcium uptake by microinjection of diaminopentane pentammine cobalt (DAPPAC), a novel inhibitor of the mitochondrial calcium uniporter. These observations demonstrate that focal SR calcium release results in calcium microdomains sufficient to promote local mitochondrial calcium uptake, suggesting a tight coupling of calcium signaling between SR release sites and nearby mitochondria.     

Fluctuating vs. Continuous Exposure to H2O2: The Effects on Mitochondrial Membrane Potential,Intracellular Calcium,and NF-κB in Astroglia

The effects of H₂O₂ are widely studied in cell cultures and other in vitro systems. However, such investigations are performed with the assumption that H₂O₂ concentration is constant, which may not properly reflect in vivo settings, particularly in redox-turbulent microenvironments such as mitochondria. Here we introduced and tested a novel concept of fluctuating oxidative stress. We treated C6 astroglial cells and primary astrocytes with H₂O₂, using three regimes of exposure – continuous, as well as fluctuating at low or high rate, and evaluated mitochondrial membrane potential and other parameters of mitochondrial activity – respiration, reducing capacity, and superoxide production, as well as intracellular ATP, intracellular calcium, and NF-κB activation. When compared to continuous exposure, fluctuating H₂O₂ induced a pronounced hyperpolarization in mitochondria, whereas the activity of electron transport chain appears not to be significantly affected. H₂O₂ provoked a decrease of ATP level and an increase of intracellular calcium concentration, independently of the regime of treatment. However, fluctuating H₂O₂ induced a specific pattern of large-amplitude fluctuations of calcium concentration. An impact on NF-κB activation was observed for high rate fluctuations, whereas continuous and low rate fluctuating oxidative stress did not provoke significant effects. Presented results outline the (patho)physiological relevance of redox fluctuations.