Ca2+ release from mitochondria induced by prooxidants

A variety of chemically different prooxidants causes Ca2+ release from mitochondria. The prooxidant-induced Ca2+ release occurs from intact mitochondria via a route which is physiologically relevant and may be regulated by protein ADP-ribosylation. When the released Ca2+ is excessively cycled by mitochondria they are damaged. This leads to uncoupling, a decreased ATP supply, and a decreased ability of mitochondria to retain Ca2+. Excessive Ca2+ cycling by mitochondria will deprive cells of ATP. As a result, Ca2+ ATPases of the endoplasmic (sarcoplasmic) reticulum and the plasma membrane are stopped. The rising cytosolic Ca2+ level cannot be counterbalanced due to damage of mitochondria which, under normoxic conditions, act as safety device against increased cytosolic Ca2+. It is proposed that prooxidants are toxic because they impair the ability of mitochondria to retain Ca2+.     

Oxidative stress in mitochondria: its relationship to cellular Ca2+ homeostasis, cell death, proliferation, and differentiation

A variety of chemically different prooxidants causes Ca2+ release from mitochondria. This prooxidant-induced Ca2+ release occurs from intact mitochondria via a route which is physiologically relevant and may be regulated by protein monoADP-ribosylation. When the released Ca2+ is excessively 'cycled' by mitochondria (continuously taken up and released) the inner membrane is damaged. This leads to a decreased ability of mitochondria to retain Ca2+, uncoupling of mitochondria, and an impairment of ATP synthesis, which in turn deprives the cell of the energy necessary for the proper functioning of the Ca2+ ATPases of the endoplasmic (sarcoplasmic) reticulum, the nucleus and the plasma membrane. The ensuing rise of the cytosolic Ca2+ level cannot be counterbalanced by the damaged mitochondria which, under normoxic conditions, act as a safety device against an increase of the cytosolic Ca2+ concentration. The impaired ability of mitochondria to retain Ca2+ may lead to cell death. However, there is also evidence emerging that release of Ca2+ from mitochondria may be physiologically important for cell proliferation and differentiation.     

Retention of calcium by mitochondria isolated from Ehrlich ascites tumor cells

Tightly coupled mitochondria isolated from Ehrlich ascites tumor cells accumulate and retain high concentrations of Ca²⁺ in the presence of ATP for periods up to at least 20 min at 25 °C. The presence of inorganic phosphate up to 20 mm does not prevent such Ca²⁺ retention. The tumor mitochondria accumulate Ca²⁺ in the presence of succinate as an energy source but lose the Ca²⁺ after 1–2 min. Addition of ATP (K_m approx 1 mm) to the incubation medium after Ca²⁺ release, induces reaccumulation of the ion. Thus, the ability of the tumor mitochondria to retain Ca²⁺ differs markedly from that of rat liver mitochondria and is seen as being of potential biological significance to the unique metabolic behavior of the ascites tumor cells.     

The role of Ca signaling in the coordination of mitochondrial ATP production with cardiac work

The heart is capable of balancing the rate of mitochondrial ATP production with utilization continuously over a wide range of activity. This results in a constant phosphorylation potential despite a large change in metabolite turnover. The molecular mechanisms responsible for generating this energy homeostasis are poorly understood. The best candidate for a cytosolic signaling molecule reflecting ATP hydrolysis is Ca²⁺. Since Ca²⁺ initiates and powers muscle contraction as well as serves as the primary substrate for SERCA, Ca²⁺ is an ideal feed-forward signal for priming ATP production. With the sarcoplasmic reticulum to cytosolic Ca²⁺ gradient near equilibrium with the free energy of ATP, cytosolic Ca²⁺ release is exquisitely sensitive to the cellular energy state providing a feedback signal. Thus, Ca²⁺ can serve as a feed-forward and feedback regulator of ATP production. Consistent with this notion is the correlation of cytosolic and mitochondrial Ca²⁺ with work in numerous preparations as well as the localization of mitochondria near Ca²⁺ release sites. How cytosolic Ca²⁺ signaling might regulate oxidative phosphorylation is a focus of this review. The relevant Ca²⁺ sensitive sites include several dehydrogenases and substrate transporters together with a post-translational modification of F1-FO-ATPase and cytochrome oxidase. Thus, Ca²⁺ apparently activates both the generation of the mitochondrial membrane potential as well as utilization to produce ATP. This balanced activation extends the energy homeostasis observed in the cytosol into the mitochondria matrix in the never resting heart.     

Effect of prooxidants on yeast mitochondria

Tightly coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts were used in this study. The two yeasts are aerobes containing the fully competent respiratory chain with three energy conservation sites. Interaction of the yeast mitochondria with prooxidants (diamide, menadione, oxaloacetate, phenylarsine oxide, hydrogen peroxide, t-butyl peroxide, and ascorbate plus Fe²⁺) was studied. The prooxidants, depending on their chemical nature, either caused uncoupling (e.g., activated state 4 respiration) or inhibited oxidation of respiratory substrates. All of the agents dissipated the membrane potential without megachannel formation (no large-scale swelling of mitochondria was observed). Except for combined application of ascorbate and Fe²⁺, the prooxidant-induced decrease in the membrane potential was specifically prevented by ATP, even in the cases when classic antioxidants, e.g., N-acetylcysteine, were ineffective. No permeabilization of yeast mitochondria was observed under concerted action of prooxidants and Ca²⁺, suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with Ca²⁺ uptake.     

Diversity of mitochondrial Ca signaling in rat neonatal cardiomyocytes: Evidence from a genetically directed Ca probe,mitycam-E31Q

I_Ca-gated Ca²⁺ release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca²⁺, but the extent to which mitochondria contribute to the overall Ca²⁺ signaling remains controversial. To examine the possible role of mitochondria in Ca²⁺ signaling, we developed a low affinity mitochondrial Ca²⁺ probe, mitycam-E31Q (300–500 MOI, 48–72 h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca²⁺ in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca²⁺ while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca²⁺) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na⁺ increased global cytosolic and mitochondrial Ca²⁺ signals in one population of mitochondria, but unexpectedly decreased it (release of Ca²⁺) in another mitochondrial population. Such mitochondrial Ca²⁺ release signals were seen not only during long lasting Na⁺ withdrawal, but also when Ca²⁺ loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca²⁺ transients appear to activate with a delay following the cytosolic rise of Ca²⁺ and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca²⁺ signaling.     

Intestinal secretagogues increase cytosolic free Ca2+ concentration and K+ conductance in a human intestinal epithelial cell line

Summary A human intestinal epithelial cell line (Intestine 407) is known to retain receptors for intestinal secretagogues such as acetylcholine (ACh), histamine, serotonin (5-HT) and vasoactive intestinal peptide (VIP). The cells were also found to possess separate receptors for secretin and ATP, the stimulation of which elicited transient hyperpolarizations coupled to decreased membrane resistances. These responses were reversed in polarity at the K⁺ equilibrium potential. The hyperpolarizing responses to six agonists were reversibly inhibited by quinine or quinidine. By means of Ca²⁺-selective microelectrodes, increases in the cytosolic free Ca²⁺ concentration were observed in response to individual secretagogues. The time course of Ca²⁺ responses coincided with that of hyperpolarizing responses. The responses to ACh and 5-HT were abolished by a reduction in the extracellular Ca²⁺ concentration down to pCa 7 or by application of Co²⁺. Thus, in Intestine 407 cells, not only the intestinal secretagogues, which are believed to act via increased cytosolic Ca²⁺ (ACh, 5-HT and histamine), but also those which elevate cyclic AMP (VIP, secretin and ATP) induce increases in cytosolic Ca²⁺, thereby activating the K⁺ conductance. It is likely that the origin of increased cytosolic Ca²⁺ is mainly extracellular for ACh- and 5-HT-induced responses, whereas histamine, VIP, secretin and ATP mobilize Ca²⁺ from the internal compartment.     

Permeability transition pore of the inner mitochondrial membrane can operate in two open states with different selectivities

Prooxidants induce release of Ca²⁺ from mitochondria through the giant solute pore in the mitochondrial inner membrane. However, under appropriate conditions prooxidants can induce Ca²⁺ release without inducing a nonspecific permeability change. Prooxidant-induced release of Ca²⁺ isselective. Presumably, this is the result of the operation of a permeability pathway for H⁺ coupled to the reversal of the Ca²⁺ uniporter, the latter generating the selectivity. The solute pore and prooxidant-induced Ca²⁺-specific pathways exhibit common sensitivities to a set of inhibitors and activators. It is proposed that the pore can operate in two open states: (1) permeable to H⁺ only and (2) permeable to solutes of M_r<1500. Under some conditions, prooxidants induce the H⁺-selective state which, in turn, collapses the inner membrane potential and permits selective loss of Ca²⁺ via the Ca²⁺ uniporter.     

Reduced capacity of Ca<Superscript>2+</Superscript> retention in liver as compared to kidney mitochondria. ADP requirement

Ca²⁺ loading in mitochondria promotes the opening of a non-selective transmembrane pathway. Permeability transition is also associated with the interaction of cyclophilin D at the internal surface of the non-specific transmembrane pore. This interaction is circumvented by cyclosporin A and ADP. Our results show that, in the absence of ADP, liver mitochondria were unable to retain Ca²⁺, they underwent a fast and large amplitude swelling, as well as a rapid collapse of the transmembrane potential. In contrast, in the absence of ADP, kidney mitochondria retained Ca²⁺, swelling did not occur, and the collapse of the membrane potential was delayed. Ca²⁺ efflux was reversed by the addition of ADP and cyclosporin A. Our findings indicate that the differences between liver and kidney mitochondria are due to the low association of cyclophilin D to the ADP/ATP carrier found in kidney mitochondria as compared to liver mitochondria.     

Calcium uptake mechanisms of mitochondria

The ability of mitochondria to capture Ca²⁺ ions has important functional implications for cells, because mitochondria shape cellular Ca²⁺ signals by acting as a Ca²⁺ buffer and respond to Ca²⁺ elevations either by increasing the cell energy supply or by triggering the cell death program of apoptosis. A mitochondrial Ca²⁺ channel known as the uniporter drives the rapid and massive entry of Ca²⁺ ions into mitochondria. The uniporter operates at high, micromolar cytosolic Ca²⁺ concentrations that are only reached transiently in cells, near Ca²⁺ release channels. Mitochondria can also take up Ca²⁺ at low, nanomolar concentrations, but this high affinity mode of Ca²⁺ uptake is not well characterized. Recently, leucine-zipper-EF hand-containing transmembrane region (Letm1) was proposed to be an electrogenic 1:1 mitochondrial Ca²⁺/H⁺ antiporter that drives the uptake of Ca²⁺ into mitochondria at nanomolar cytosolic Ca²⁺ concentrations. In this article, we will review the properties of the Ca²⁺ import systems of mitochondria and discuss how Ca²⁺ uptake via an electrogenic 1:1 Ca²⁺/H⁺ antiport challenges our current thinking of the mitochondrial Ca²⁺ uptake mechanism.     

Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: Mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store

Mitochondrial Ca²⁺ plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca²⁺ imaging is an important approach to understand mitochondrial Ca²⁺ dynamics. Recently developed GCaMP genetically-encoded Ca²⁺ indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca²⁺ imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca²⁺ dynamics in culture, revealing Ca²⁺ uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca²⁺ indicators, our results show that mitochondrial Ca²⁺ uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca²⁺ dynamics on cytosolic Ca²⁺ changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca²⁺ increase using mito-GCaMP5G and a synthetic organic Ca²⁺ indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca²⁺ signaling pathway, our results demonstrated that the mitochondrial Ca²⁺ uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca²⁺ release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca²⁺ dynamics as well as Ca²⁺ interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease.     

Mitochondrial dynamics in human NADH:ubiquinone oxidoreductase deficiency

Mitochondrial NADH:ubiquinone oxidoreductase or complex I (CI) is a frequently affected enzyme in cases of mitochondrial disorders. However, the cytopathological mechanism of the associated pediatric syndromes is poorly understood. Evidence in the literature suggests a connection between mitochondrial metabolism and morphology. Previous quantitative analysis of mitochondrial structure in cultured fibroblasts of 14 patients revealed that mitochondria were fragmented and/or less branched in patients with severe CI deficiency. These patient cells also displayed greatly increased levels of reactive oxygen species (ROS) and marked aberrations in mitochondrial and cellular Ca²⁺/ATP handling upon hormone stimulation. Here, we discuss the interrelationship between these parameters and demonstrate that the hormone-induced increase in mitochondrial Ca²⁺ and ATP concentration, as well as the rate of cytosolic Ca²⁺ removal, are not related to mitochondrial length and/or degree of branching, but decrease as a function of the number of mitochondria per cell. This suggests that the amount of mitochondria, and not their shape, is important for Ca²⁺-induced stimulation of mitochondrial ATP generation to feed cytosolic ATP-demanding processes.     

Mitochondrial Ca2+ Uptake 1 (MICU1) and Mitochondrial Ca2+ Uniporter (MCU) Contribute to Metabolism-Secretion Coupling in Clonal Pancreatic ��-Cells

In pancreatic β-cells, uptake of Ca²⁺ into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca²⁺ sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca²⁺ uptake 1 (MICU1), mitochondrial Ca²⁺ uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca²⁺ sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca²⁺ uptake upon both intracellular Ca²⁺ release and Ca²⁺ entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca²⁺ uptake in response to either intracellular Ca²⁺ release or Ca²⁺ entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca²⁺ uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca²⁺ oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca²⁺ uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.     

Interaction of calcium with mitochondria isolated from Ehrlich ascites tumour cells

Calcium ions are accumulated by intact mitochondria isolated from Ehrlich ascites tumour cells in a buffered system supplemented with ATP or succinate. In the ATP-supplemented system, the tumour mitochondria, in contrast to rat liver mitochondria, retain the accumulated Ca²⁺, do not exhibit a marked “irreversible” ATPase and do not swell. In the succinate-supplemented system, added Ca²⁺ stimulates respiration in either the absence or presence of added inorganic phosphate. Whereas respiration by rat liver mitochondria, measured in the presence of added phosphate, remains continuously activated after the addition of only a small amount of Ca²⁺, that by the tumour mitochondria can be stimulated by several successive additions of 100 μM Ca²⁺ and at all times exhibit appreciable activation ratios.     

Control of the pyridine nucleotide-linked Ca release from mitochondria by respiratory substrates

Oxidation of mitochondrial pyridine nucleotides followed by their hydrolysis promotes Ca²⁺ release from intact liver mitochondria. In most of the previous studies oxidation was achieved with pro-oxidants which were added to mitochondria respiring on succinate in the presence of rotenone, a site I-specific inhibitor of the respiratory chain. Here we investigate pro-oxidant dependent and independent Ca²⁺ release from mitochondria when respiration is supported either by the NAD⁺-linked substrate β-hydroxybutyrate, or by succinate. In the presence, as well as in the absence, of the pro-oxidant t-butylhydroperoxide mitochondria retain Ca²⁺ much better with succinate than with β-hydroxybutyrate, as respiratory substrate. When Ca²⁺ release is induced by t-butylhydroperoxide succinate-supported Ca²⁺ retention is impeded by rotenone. Ca²⁺ release (pro-oxidant dependent or independent) is paralleled by oxidation and hydrolysis of intramitochondrial pyridine nucleotides, and Ca²⁺ retention is paralleled by reduction of pyridine nucleotides. It is concluded that the pyridine nucleotide-linked Ca²⁺ release from mitochondria can be controlled by respiratory substrates which regulate the intramitochondrial hydrolysis of oxidized pyridine nucleotides.     

Ca2+ Homeostasis in the Agonist-sensitive Internal Store: Functional Interactions Between Mitochondria and the ER Measured In Situ in Intact Cells

Mitochondria have a well-established capacity to detect cytoplasmic Ca²⁺ signals resulting from the discharge of ER Ca²⁺ stores. Conversely, both the buffering of released Ca²⁺ and ATP production by mitochondria are predicted to influence ER Ca²⁺ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca²⁺] directly using trapped fluorescent low-affinity Ca²⁺ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca²⁺ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca²⁺ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca²⁺ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca²⁺ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca²⁺ signals.     

Mitochondria Regulate Neutrophil Activation by Generating ATP for Autocrine Purinergic Signaling

Polymorphonuclear neutrophils (PMNs) form the first line of defense against invading microorganisms. We have shown previously that ATP release and autocrine purinergic signaling via P2Y₂ receptors are essential for PMN activation. Here we show that mitochondria provide the ATP that initiates PMN activation. Stimulation of formyl peptide receptors increases the mitochondrial membrane potential (Δψm) and triggers a rapid burst of ATP release from PMNs. This burst of ATP release can be blocked by inhibitors of mitochondrial ATP production and requires an initial formyl peptide receptor-induced Ca²⁺ signal that triggers mitochondrial activation. The burst of ATP release generated by the mitochondria fuels a first phase of purinergic signaling that boosts Ca²⁺ signaling, amplifies mitochondrial ATP production, and initiates functional PMN responses. Cells then switch to glycolytic ATP production, which fuels a second round of purinergic signaling that sustains Ca²⁺ signaling via P2X receptor-mediated Ca²⁺ influx and maintains functional PMN responses such as oxidative burst, degranulation, and phagocytosis.     

OSMOTICALLY LYSED RAT LIVER MITOCONDRIA : Biochemical and Ultrastructural Properties in Relation to Massive Ion Accumulation

Osmotically lysed rat liver mitochondria have been utilized for a study of the biochemical and ultrastructural properties in relation to divalent ion accumulation. Osmotic lysis of mitochondria by suspension and washing in cold, distilled water results in the extraction of about 50% of the mitochondrial protein, the loss of the outer mitochondrial membrane, an increase in respiration, and a marked decrease in the ability to catalyze oxidative phosphorylation. Nevertheless, except for a decrease in the ability to accumulate Sr²⁺ by an ATP-supported process, these lysed mitochondria retain full capacity to accumulate massive amounts of divalent cations by respiration-dependent and ATP-supported mechanisms. The decreased ability of osmotically lysed mitochondria to accumulate Sr²⁺ by an ATP-energized process does not appear to be due to a loss or inactivation of a specific Sr²⁺-activated ATPase. The energy-dependent accumulation processes in lysed mitochondria show an increased sensitivity to inhibition by monovalent cations. Extraction of cytochrome c from osmotically lysed mitochondria results in a complete loss of phosphorylation and the respiration-dependent accumulation of Ca²⁺; a lesser, but significant, decrease in the ATP-supported accumulation of Ca²⁺ also was observed. The addition of cytochrome c fully restores the respiration-dependent accumulation of Ca²⁺ to the level present in unextracted, osmotically lysed mitochondria. The ATP-supported process is not affected by the addition of cytochrome c to extracted mitochondria, indicating that cytochrome c is not involved in ion transport energized by ATP. The osmotically lysed mitochondria are devoid of outer membranes and contain relatively little matrix substance. The accumulation of Ca²⁺ and P_i by lysed mitochondria under massive loading conditions is accompanied by the formation of electron-opaque deposits within the lysed mitochondria associated with the inner membranes. This finding suggests that the inner membrane plays a role in the deposition of divalent ions within intact rat liver mitochondria. The relevance of these observations to those of other investigators is discussed.     

Stable enhancement of calcium retention in mitochondria isolated from rat liver after the administration of glucagon to the intact animal

1. Mitochondria isolated from rat liver by centrifugation of the homogenate in buffered iso-osmotic sucrose at between 4000 and 8000g-min, 1h after the administration in vivo of 30μg of glucagon/100g body wt., retain Ca²⁺ for over 45min after its addition at 100nmol/mg of mitochondrial protein in the presence of 2mm-P_i. In similar experiments, but after the administration of saline (0.9% NaCl) in place of glucagon, Ca²⁺ is retained for 6–8min. The ability of glucagon to enhance Ca²⁺ retention is completely prevented by co-administration of 4.2mg of puromycin/100g body wt. 2. The resting rate of respiration after Ca²⁺ accumulation by mitochondria from glucagon-treated rats remains low by contrast with that from saline-treated rats. Respiration in the latter mitochondria increased markedly after the Ca²⁺ accumulation, reflecting the uncoupling action of the ion. 3. Concomitant with the enhanced retention of Ca²⁺ and low rates of resting respiration by mitochondria from glucagon-treated rats was an increased ability to retain endogenous adenine nucleotides. 4. An investigation of properties of mitochondria known to influence Ca²⁺ transport revealed a significantly higher concentration of adenine nucleotides but not of P_i in those from glucagon-treated rats. The membrane potential remained unchanged, but the transmembrane pH gradient increased by approx. 10mV, indicating increased alkalinity of the matrix space. 5. Depletion of endogenous adenine nucleotides by P_i treatment in mitochondria from both glucagon-treated and saline-treated rats led to a marked diminution in ability to retain Ca²⁺. The activity of the adenine nucleotide translocase was unaffected by glucagon treatment of rats in vivo. 6. Although the data are consistent with the argument that the Ca²⁺-translocation cycle in rat liver mitochondria is a target for glucagon action in vivo, they do not permit conclusions to be drawn about the molecular mechanisms involved in the glucagon-induced alteration to this cycle.     

Ca sources for the exocytotic release of glutamate from astrocytes

Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca²⁺ concentration is necessary and sufficient for this process. The predominant source of Ca²⁺ for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca²⁺ to the cytosol. The ER store is (re)filled by the store-specific Ca²⁺-ATPase. Ultimately, the depleted ER is replenished by Ca²⁺ which enters from the extracellular space to the cytosol via store-operated Ca²⁺ entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca²⁺ channels and plasma membrane Na⁺/Ca²⁺ exchangers are additional means for cytosolic Ca²⁺ entry. Cytosolic Ca²⁺ levels can be modulated by mitochondria, which can take up cytosolic Ca²⁺ via the Ca²⁺ uniporter and release Ca²⁺ into cytosol via the mitochondrial Na⁺/Ca²⁺ exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca²⁺ sources generates cytosolic Ca²⁺ dynamics that can drive Ca²⁺-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.