Mitochondrial Calcium Sequestration Research Articles

Sleep/wake calcium dynamics, respiratory function, and ROS production in cardiac mitochondria

IntroductionIncidents of myocardial infarction and sudden cardiac arrest vary with time of the day, but the mechanism for this effect is not clear. We hypothesized that diurnal changes in the ability of cardiac mitochondria to control calcium homeostasis dictate vulnerability to cardiovascular events. ObjectivesHere we investigate mitochondrial calcium dynamics, respiratory function, and reactive oxygen species (ROS) production in mouse heart during different phases of wake versus sleep periods. MethodsWe assessed time-of-the-day dependence of calcium retention capacity of isolated heart mitochondria from young male C57BL6 mice. Rhythmicity of mitochondrial-dependent oxygen consumption, ROS production and transmembrane potential in homogenates were explored using the Oroboros O2k Station equipped with a fluorescence detection module. Changes in expression of essential clock and calcium dynamics genes/proteins were also determined at sleep versus wake time points. ResultsOur results demonstrate that cardiac mitochondria exhibit higher calcium retention capacity and higher rates of calcium uptake during sleep period. This was associated with higher expression of clock gene Bmal1, lower expression of per2, greater expression of MICU1 gene (mitochondrial calcium uptake 1), and lower expression of the mitochondrial transition pore regulator gene cyclophilin D. Protein levels of mitochondrial calcium uniporter (MCU), MICU2, and sodium/calcium exchanger (NCLX) were also higher at sleep onset relative to wake period. While complex I and II-dependent oxygen utilization and transmembrane potential of cardiac mitochondria were lower during sleep, ROS production was increased presumably due to mitochondrial calcium sequestration. ConclusionsTaken together, our results indicate that retaining mitochondrial calcium in the heart during sleep dissipates membrane potential, slows respiratory activities, and increases ROS levels, which may contribute to increased vulnerability to cardiac stress during sleep-wake transition. This pronounced daily oscillations in mitochondrial functions pertaining to stress vulnerability may at least in part explain diurnal prevalence of cardiac pathologies.

Mitochondrial ultrastructure, function, and calcium buffering by massive calcium loading observed using advanced cryo‐electron microscopy, high‐resolution respirometry and spectrofluorimetry

Excessive calcium accumulation is the main cause of cardiac tissue and cell death after a myocardial infarction. As a result of calcium overload, mitochondria become dysfunctional, produce excessive reactive oxygen species (ROS), cannot support the cellular energetic demand, and activate the cyclosporine A‐sensitive mitochondrial permeability transition pore. To discern the molecular, bioenergetic, and structural events associated with this phenomenon, we collected and analyzed a time‐course series of images obtained by utilizing recent advances in electron cryo‐microscopy methods. Energized mitochondria exposed to high calcium boluses, sufficient to elicit mitochondrial permeability transition, resulted in the formation of calcium phosphate granules. Under high calcium loading, the size of the calcium‐phosphate granules ranged from 50–120 nm causing OMM/IMM rupture. CsA treatment resulted in mitochondria with aberrant morphologies, increased cristae density and number, and with larger calcium‐phosphate granules ranging from 40–200 nm. The OMM was partially or completely lost; however, the mitochondria were fully functional and capable of synthesizing ATP. Our tomographic reconstruction placed these granules in close vicinity to the IMM and cristae. In addition, CsA treatment increases the calcium buffering power at greater calcium concentrations, whereas untreated mitochondria had much lower calcium buffering. Our results demonstrate that these granules form an intricate part of the mitochondrial calcium sequestration system. Overall, these results suggest CsA may interact with IMM components to: 1) modulate calcium buffering by facilitating more abundant and larger calcium phosphate granules and 2) preserve the cristae shape and number which avoids cytochrome c loss and mitochondrial permeability transition. Our data reveal that drugs capable of modulating cristae shape and number may be an effective strategy to prevent mitochondrial dysfunction stemming from calcium overload.Support or Funding InformationThis work was supported by NIH grant R00‐HL121160.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Measurement and Analysis of the Mitochondrial Calcium Sequestration and Effects on the Buffering System

Calcium is a key signaling molecule found to regulate many cellular processes, including but not limited to, cellular physiology, bioenergetics, transcriptional events, signal transduction, and reactive oxygen species (ROS) production. While mitochondrial calcium dynamics have been extensively studied, it is yet unclear how calcium is sequestered and buffered in a quantitatively and mechanistic manner. Mitochondrial‐calcium homeostasis plays a crucial role in cell fate by a tightly‐regulated buffering system that maintains sub‐lethal levels of free calcium. If the calcium buffering capacity is exceeded mitochondrial dysfunction can result in excessive ROS production, initiating cell death signaling pathways, and catastrophic energy failure. In this study, we quantitatively characterized the mitochondrial calcium buffering power under a variety of experimental conditions and two calcium loading protocols. This was done with isolated cardiac mitochondria from guinea pigs using calcium‐sensitive molecular dyes. The mitochondrial calcium buffering power was quantified by monitoring the calcium flux from both outside and inside the mitochondria. Here, we report the changes in calcium buffering power caused by oxidative stress and under conditions that promote calcium uptake and retention. Furthermore, we demonstrate that the buffering power is also affected by the way calcium is loaded into the mitochondria. These data provide further insights on the mitochondrial calcium loading capacity and raises the question as to what are the components that regulate free calcium in the system.Support or Funding InformationThis research is supported by the National Heart, Lung, and Blood Institute (R00‐HL121160).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages

Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr-/- mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis invivo.

Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids.

Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψm) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψm in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis.

Synergistic effects of ascorbate and sorafenib in hepatocellular carcinoma: New insights into ascorbate cytotoxicity

We investigated the mechanism of selective ascorbate-induced cytotoxicity in tumor cells, including Hep G2 cells, compared to primary hepatocytes. H2O2 formation was required for ascorbate cytotoxicity, as extracellular catalase treatment protected tumor cells. H2O2 generated by glucose oxidase treatment also caused cell killing, but treatment with a pharmacologic dose (5–20mM) of ascorbate was significantly more cytotoxic at comparable rates of H2O2 production, suggesting that ascorbate enhanced H2O2 cytotoxicity. This was further supported by the finding that ascorbate at a non-cytotoxic dose (1mM) enhanced cell killing caused by glucose oxidase. Consistent with this conclusion, ascorbate treatment caused deregulation of cellular calcium homeostasis, resulting in massive mitochondrial calcium accumulation. Ascorbate acted synergistically with the chemotherapeutic sorafenib in killing Hep G2 cells, but not primary hepatocytes, suggesting adjuvant ascorbate treatment can broaden sorafenib’s therapeutic range. Sorafenib caused mitochondrial depolarization and prevented mitochondrial calcium sequestration. Subsequent ascorbate addition further deregulated cellular calcium homeostasis promoting cell death. Additionally, we present the case of a patient with hepatocellular carcinoma (HCC) who had prolonged regression of a rib metastasis upon combination treatment with ascorbate and sorafenib, indicating that these studies have direct clinical relevance.

Mitochondria in Hypoxic Pulmonary Vasoconstriction

Mitochondria in Hypoxic Pulmonary Vasoconstriction

Calcium‐dependent mitochondrial function and dysfunction in neurons

Calcium is an extraordinarily versatile signaling ion, encoding cellular responses to a wide variety of external stimuli. In neurons, mitochondria can accumulate enormous amounts of calcium, with the consequence that mitochondrial calcium uptake, sequestration and release play pivotal roles in orchestrating calcium-dependent responses as diverse as gene transcription and cell death. In this review, we consider the basic chemistry of calcium as a 'sticky' cation, which leads to extremely high bound/free ratios, and discuss areas of current interest or controversy. Topics addressed include methodologies for measuring local intracellular calcium, mitochondrial calcium buffering and loading capacity, mitochondrially directed spatial calcium gradients, and the role of calcium overload-dependent mitochondrial dysfunction in glutamate-evoked excitotoxic injury and neurodegeneration. Finally, we consider the relationship between delayed calcium de-regulation, the mitochondrial permeability transition and the generation of reactive oxygen species, and propose a unified view of the 'source specificity' and 'calcium overload' models of N-methyl-d-aspartate (NMDA) receptor-dependent excitotoxicity. Non-NMDA receptor mechanisms of excitotoxicity are discussed briefly.

Mitochondrial calcium sequestration and protein kinase C cooperate in the regulation of cortical F‐actin disassembly and secretion in bovine chromaffin cells

Mitochondria play an important role in the homeostasis of intracellular Ca(2+) and regulate its availability for exocytosis. Inhibitors of mitochondria Ca(2+) uptake such as protonophore CCCP potentiate the secretory response to a depolarizing pulse of K(+). Exposure of cells to agents that directly (cytochalasin D, latrunculin B) or indirectly (PMA) disrupt cortical F-actin networks also potentiate the secretory response to high K(+). The effects of cytochalasin D and CCCP on secretion were additive whereas those of PMA and CCCP were not; this suggests different mechanisms for cytochalasin D and CCCP and a similar mechanism for PMA and CCCP. Mitochondria were the site of action of CCCP, because the potentiation of secretion by CCCP was observed even after depletion of Ca(2+) from the endoplasmic reticulum. CCCP induced a small increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that was not modified by the protein kinase C (PKC) inhibitor chelerythrine. Both CCCP and PMA induced cortical F-actin disassembly, an effect abolished by chelerythrine. In addition, rotenone and oligomycin A, two other mitochondrial inhibitors, also evoked cortical F-actin disassembly and potentiated secretion; again, these effects were blocked by chelerythrine. CCCP also enhanced the phosphorylation of PKC and myristoylated alanine-rich C kinase substance (MARCKS), and these were also inhibited by chelerythrine. The results suggest that the rapid sequestration of Ca(2+) by mitochondria would protect the cell from an enhanced PKC activation and cortical F-actin disassembly, thereby limiting the magnitude of the secretory response.

Iron toxicity and chelation therapy.

Iron is an essential mineral for normal cellular physiology, but an excess can result in cell injury. Iron in low-molecular-weight forms may play a catalytic role in the initiation of free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates; the result is wide-ranging impairment in cellular function and integrity. The rate of free radical production must overwhelm the cytoprotective defenses of cells before injury occurs. There is substantial evidence that iron overload in experimental animals can result in oxidative damage to lipids in vivo, once the concentration of iron exceeds a threshold level. In the liver, this lipid peroxidation is associated with impairment of membrane-dependent functions of mitochondria and lysosomes. Iron overload impairs hepatic mitochondrial respiration primarily through a decrease in cytochrome C oxidase activity, and hepatocellular calcium homeostasis may be compromised through damage to mitochondrial and microsomal calcium sequestration. DNA has also been reported to be a target of iron-induced damage, and this may have consequences in regard to malignant transformation. Mitochondrial respiratory enzymes and plasma membrane enzymes such as sodium-potassium-adenosine triphosphatase (Na(+) + K(+)-ATPase) may be key targets of damage by non-transferrin-bound iron in cardiac myocytes. Levels of some antioxidants are decreased during iron overload, a finding suggestive of ongoing oxidative stress. Reduced cellular levels of ATP, lysosomal fragility, impaired cellular calcium homeostasis, and damage to DNA all may contribute to cellular injury in iron overload. Evidence is accumulating that free-radical production is increased in patients with iron overload. Iron-loaded patients have elevated plasma levels of thiobarbituric acid reactants and increased hepatic levels of aldehyde-protein adducts, indicating lipid peroxidation. Hepatic DNA of iron-loaded patients shows evidence of damage, including mutations of the tumor suppressor gene p53. Although phlebotomy therapy is effective in removing excess iron in hereditary hemochromatosis, chelation therapy is required in the treatment of many patients who have combined secondary and transfusional iron overload due to disorders in erythropoiesis. In patients with beta-thalassemia who undergo regular transfusions, deferoxamine treatment has been shown to be effective in preventing iron-induced tissue injury and in prolonging life expectancy. The use of the oral chelator deferiprone remains controversial, and work is continuing on the development of new orally effective iron chelators.

Presynaptic mitochondrial calcium sequestration influences transmission at mammalian central synapses.

Beyond their role in generating ATP, mitochondria have a high capacity to sequester calcium. The interdependence of these functions and limited access to presynaptic compartments makes it difficult to assess the role of sequestration in synaptic transmission. We addressed this important question using the calyx of Held as a model glutamatergic synapse by combining patch-clamp with a novel mitochondrial imaging method. Presynaptic calcium current, mitochondrial calcium concentration ([Ca(2+)](mito), measured using rhod-2 or rhod-FF), cytoplasmic calcium concentration ([Ca(2+)](cyto), measured using fura-FF), and the postsynaptic current were monitored during synaptic transmission. Presynaptic [Ca(2+)](cyto) rose to 8.5 +/- 1.1 microM and decayed rapidly with a time constant of 45 +/- 3 msec; presynaptic [Ca(2+)](mito) also rose rapidly to >5 microM but decayed slowly with a half-time of 1.5 +/- 0.4 sec. Mitochondrial depolarization with rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone abolished mitochondrial calcium rises and slowed the removal of [Ca(2+)](cyto) by 239 +/- 22%. Using simultaneous presynaptic and postsynaptic patch clamp, combined with presynaptic mitochondrial and cytoplasmic imaging, we investigated the influence of mitochondrial calcium sequestration on transmitter release. Depletion of ATP to maintain mitochondrial membrane potential was blocked with oligomycin, and ATP was provided in the patch pipette. Mitochondrial depolarization raised [Ca(2+)](cyto) and reduced transmitter release after short EPSC trains (100 msec, 200 Hz); this effect was reversed by raising mobile calcium buffering with EGTA. Our results suggest a new role for presynaptic mitochondria in maintaining transmission by accelerating recovery from synaptic depression after periods of moderate activity. Without detectable thapsigargin-sensitive presynaptic calcium stores, we conclude that mitochondria are the major organelle regulating presynaptic calcium at central glutamatergic terminals.

Presynaptic Mitochondrial Calcium Sequestration Influences Transmission at Mammalian Central Synapses

Beyond their role in generating ATP, mitochondria have a high capacity to sequester calcium. The interdependence of these functions and limited access to presynaptic compartments makes it difficult to assess the role of sequestration in synaptic transmission. We addressed this important question using the calyx of Held as a model glutamatergic synapse by combining patch-clamp with a novel mitochondrial imaging method. Presynaptic calcium current, mitochondrial calcium concentration ([Ca2+]mito, measured using rhod-2 or rhod-FF), cytoplasmic calcium concentration ([Ca2+]cyto, measured using fura-FF), and the postsynaptic current were monitored during synaptic transmission. Presynaptic [Ca2+]cytorose to 8.5 ± 1.1 μmand decayed rapidly with a time constant of 45 ± 3 msec; presynaptic [Ca2+]mitoalso rose rapidly to >5 μmbut decayed slowly with a half-time of 1.5 ± 0.4 sec. Mitochondrial depolarization with rotenone and carbonyl cyanidep-trifluoromethoxyphenylhydrazone abolished mitochondrial calcium rises and slowed the removal of [Ca2+]cytoby 239 ± 22%. Using simultaneous presynaptic and postsynaptic patch clamp, combined with presynaptic mitochondrial and cytoplasmic imaging, we investigated the influence of mitochondrial calcium sequestration on transmitter release. Depletion of ATP to maintain mitochondrial membrane potential was blocked with oligomycin, and ATP was provided in the patch pipette. Mitochondrial depolarization raised [Ca2+]cytoand reduced transmitter release after short EPSC trains (100 msec, 200 Hz); this effect was reversed by raising mobile calcium buffering with EGTA. Our results suggest a new role for presynaptic mitochondria in maintaining transmission by accelerating recovery from synaptic depression after periods of moderate activity. Without detectable thapsigargin-sensitive presynaptic calcium stores, we conclude that mitochondria are the major organelle regulating presynaptic calcium at central glutamatergic terminals.

Metal-induced hepatotoxicity.

Figure 3 summarizes several proposed mechanisms of iron- or copper-induced hepatotoxicity. It has long been suspected that free radicals may play a role in iron- and copper-induced cell toxicity because of the powerful prooxidant action of iron and copper salts in vitro. In the presence of available cellular reductants, iron or copper in low molecular weight forms may play a catalytic role in the initiation of free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates, resulting in wide-ranging impairment in cellular function and integrity. However, cells are endowed with cytoprotective mechanisms (antioxidants, scavenging enzymes, repair processes) that act to counteract the effects of free radical production. Thus, the net effect of metal-induced free radicals on cellular function will depend on the balance between radical production and the cytoprotective systems As a result, there may be a rate of free radical production that must be exceeded before cellular injury occurs. Evidence has now accumulated that iron or copper overload in experimental animals can result in oxidative damage to lipids in vivo, once the concentration of the metal exceeds a threshold level. In the liver, this lipid peroxidation is associated with impairment of membrane-dependent functions of mitochondria (oxidative metabolism) and lysosomes (membrane integrity, fluidity, pH). Although these findings do not prove causality, it seems likely that lipid peroxidation is involved, since similar functional defects are produced by metal-induced lipid peroxidation in these organelles in vitro. Both iron and copper overload impair hepatic mitochondrial respiration, primarily through a decrease in cytochrome c oxidase activity. In iron overload, hepatocellular calcium homeostasis may be impaired through damage to mitochondrial and microsomal calcium sequestration. DNA has also been reported to be a target of metal-induced damage in the liver; this may have consequences as regards malignant transformation. The levels of some antioxidants in the liver are decreased in rats with iron or copper overload, which is also suggestive of ongoing oxidative stress. Reduced cellular ATP levels, lysosomal fragility, impaired cellular calcium homeostasis, and damage to DNA may all contribute to hepatocellular injury in iron and copper overload. There are few data addressing the key issue of whether free radical production is increased in patients with iron or copper overload. Patients with hereditary hemochromatosis have elevated plasma levels of TBA-reactants and increased hepatic levels of MDA-protein and HNE-protein adducts, indicative of lipid peroxidation. Mitochondria isolated from the livers of Wilson disease patients have evidence of lipid peroxidation, and some patients with Wilson disease have decreased hepatic and plasma levels of vitamin E. Additional investigation will be required to fully assess oxidant stress and its potential pathophysiologic role in patients with iron or copper overload.

Ultrastructural localization of calcium in ischemic hippocampal slices: the influence of adenosine and theophylline.

Calcium was localized ultrastructurally with the use of the modified oxalate-pyroantimonate reaction in the CA1 region of rat hippocampal slices. Ten-minute ischemia (incubation with anoxic and glucose-free medium) followed by 30 min reoxygenation resulted in mitochondrial calcium sequestration and ultrastructural damage. The addition of the adenosine receptor antagonist, theophylline, worsened the ischemia-induced morphological changes and particularly exaggerated the Ca2+ loading in the postsynaptic dendrites. In contrast, adenosine protected against ischemia-induced changes. The results suggest that adenosine exerts its neuroprotective action largely by maintaining intracellular calcium-homeostasis.

Chronic dietary iron overload in rats results in impaired calcium sequestration by hepatic mitochondria and microsomes 1, 2, and 3

The purpose of these experiments was to determine whether chronic dietary iron overload causes impairment of hepatic mitochondrial and/or microsomal calcium sequestration. Experimental iron overload was produced by feeding three groups of rats a chow diet supplemented with 3.0% (wt/wt) carbonyl iron for up to 8 weeks achieving graded increases in hepatic iron concentrations ranging from 1360 to 3170 μg/g. At low levels of iron overload, there were no changes in mitochondrial oxidative metabolism or calcium sequestration, whereas at moderate and high degrees of iron loading, both of these parameters were significantly reduced. In contrast, there were significant decreases in microsomal cytochrome P450 levels and microsomal calcium sequestration at all three levels of iron loading. These abnormalities occurred at hepatic iron concentrations at which the authors have previously found evidence of hepatic organelle lipid peroxidation. These alterations in organelle calcium sequestration may impair intracellular calcium homeostasis in the liver and contribute to subsequent cellular injury.

Effect of Oxalate on Function of Kidney Mitochondria

The effects of oxalate on kidney mitochondria were evaluated in vitro to test whether oxalate exposure leads to derangement(s) in mitochondrial function that could in turn promote the formation of kidney stones. Our previous studies demonstrated that oxalate is transported across the mitochondrial membrane via the dicarboxylate carrier. The present studies indicated that oxalate competitively inhibits the uptake and oxidation of exogenous malate and succinate in isolated mitochondria but has no effect on mitochondrial respiration in the presence of a mixture of glutamate plus malate or glutamate plus pyruvate. Oxalate attenuates the increase in mitochondrial respiration produced by the uncoupler CCCP or by the Ca2+ ionophore A23187, and the latter effect is more pronounced in kidney than in liver mitochondria. The apparent Ki of oxalate for the response to Ca2+ ionophore is 1.9 ± 0.3mM in kidney and 6.1 ± 0.2mM in liver mitochondria. Similarly, the ability of oxalate to attenuate calcium-induced swelling of mitochondria is more dramatic in kidney than in liver mitochondria (apparent KiS of 1.7 ± 0.1 and 18.2 ± 0.7mM, respectively). Oxalate has no effect on the rate of calcium uptake by energized mitochondria or on the rate of ruthenium red-insensitive calcium efflux from mitochondria in either tissue. The above findings indicate that oxalate interacts with the inner mitochondrial membrane or with processes controlling membrane integrity to a greater extent in kidney than liver mitochondria. The effects of oxalate on membrane permeability or integrity may be more important than its effects on mitochondrial energy production or calcium sequestration in the pathogenesis of calcium oxalate microlith formation in the kidney.

Intraendothelial accumulation of calcium in the hippocampus and thalamus of rats after systemic kainic acid administration.

The accumulation of calcium in the hippocampal and thalamic vascular endothelium and the perivascular space was detected histochemically by means of the pyroantimonate technique 30, 60 and 120 min after systemic kainic acid administration. An increased number of calcium pyroantimonate deposits was found in the endothelial mitochondria 60 min after kainate injection. The mitochondria were swollen at this time and vacuoles containing deposits were observed. After 120 min a pronounced perivascular glial swelling was conspicuous, besides the numerous endothelial mitochondrial deposits. The swollen glial processes contained a large number of pyroantimonate deposits. It seems likely that the transendothelial calcium transport processes are accompanied by intraendothelial calcium accumulation and mitochondrial calcium sequestration.

Neuronal alterations in hippocampus following severe hypoglycaemia: a light microscopic and ultrastructural study in the rat.

Because they induce similar neuropathological changes (ischaemic cell change with microvacuolization), it has been suggested that ischaemia, status epilepticus and hypoglycaemia produce cell death by similar mechanisms, especially those resulting from intracellular calcium accumulation. We have recently demonstrated microvacuolation of neurons, mitochondrial swelling (the electron microscopic correlate of microvacuolization) and massive mitochondrial calcium sequestration (using the pyroantimonate technique) following ischaemia or status epilepticus. We therefore studied the selectively vulnerable neurons of rat hippocampus by light and electron microscopy (including the pyroantimonate technique) following 30 and 60 min of EEG isoelectricity resulting from insulin hypoglycaemia. The neuropathology at the light and EM level is unique and different from that following status epilepticus or ischaemia. The most constant finding is dark cell change of the granule cells at the tip of the dentate gyrus. In contrast to status epilepticus and ischaemia, hippocampal pyramidal neurons are far less frequently involved. Microvacuoles are rarely seen and, when present, their ultrastructural correlate is swollen Golgi apparatus, not dilated mitochondria. No intracellular calcium accumulation is demonstrable with pyroantimonate technique. Thus the cellular alterations produced by hypoglycaemia differ in character and distribution from those produced by anoxia-ischaemia. Mitochondrial calcium accumulation is not prominent in cell death from hypoglycaemia. Whether calcium toxicity plays another, subtler role in hypoglycaemic brain injury is unknown.

Mitochondrial calcium sequestration in cortical and hippocampal neurons after prolonged ischemia of the cat brain.

Adult normothermic cats were submitted to 1- h complete cerebrocirculatory arrest, followed by blood recirculation for 6-8 h. Two groups of animals could be distinguished: In one group electrocorticogram and somatically evoked primary cortical potentials steadily recovered after ischemia, and in another electrophysiologic recovery was absent. At the end of the recirculation period, calcium content was measured in tissue samples taken from cerebral cortex and hippocampus, and compared with mitochondrial calcium sequestration as assessed by electron-microscopic cytochemistry. Protein content of cortex and hippocampus was also determined for evaluation of tissue swelling. The two regions were selected because previous experiments had revealed that in animals with electrophysiologic recovery cerebral cortex remains intact although hippocampus is selectively injured, whereas in animals without electrophysiologic recovery both cerebral cortex and hippocampus are damaged. In animals with functional recovery, neither calcium content nor mitochondrial calcium sequestration were significantly increased in either cerebral cortex or hippocampal subfield CA1. Only in dentate gyrus a minor degree of mitochondrial calcium sequestration was present. Calculation of tissue swelling revealed no change in cerebral cortex, but a volume increase by 18% in hippocampus, indicating development of brain edema in this region. In animals without functional recovery tissue calcium significantly increased both in cortex and hippocampus (by 49% and 73% of control, respectively), and there was significant mitochondrial calcium accumulation in both regions. Calculated brain swelling in these animals amounted to 16% and 26% in cortex and hippocampus, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of sodium, hydrogen and magnesium ions on mitochondrial calcium sequestration in adult rat ventricular myocytes.

The effect of Na+, H+ and Mg2+ ions on net calcium exchange induced in digitonin-treated myocytes has been investigated. Raising the [Na] from 1.4 to 31.4 mM revealed a sodium-sensitive fraction of net calcium exchange with a K1/2 for Na+ ions of 12 mM, alongside the respiration-dependent accumulation of calcium. An acidosis, but not an alkalosis, was found to depress both of these processes. Mg2+ ions exerted an effect solely on the respiration-dependent calcium sequestration. A simple semi-empirical model based on the experimental data was formulated to assess the effects that altering sarcoplasmic [Na+] and [H+] would have on the calcium-handling properties of cardiac mitochondria. It is concluded that part of the inotropic effects of these ions could be mediated via this organelle.