Bile Acid-Induced Ca<Superscript>2+</Superscript> Efflux from Liver Mitochondria as a Factor Preventing the Formation of Mitochondrial Pores

The effect of bile acids as inducers of Ca²⁺ efflux from the matrix was studied on isolated rat liver mitochondria. Mitochondria in the presence of cyclosporin A (CsA) were energized by succinate, then loaded with Ca²⁺ and after the addition of the calcium uniporter inhibitor ruthenium red were de-energized by malonate. It was shown that under these conditions hydrophobic bile acids lithocholic and chenodeoxycholic at concentrations of 10 and 30 μM respectively and hydrophilic bile acids ursodeoxycholic and cholic at a concentration of 400 μM induce Ca²⁺ efflux from the mitochondrial matrix. It is noted that the efflux of these ions is not associated with damage of the inner mitochondrial membrane by bile acids, since it is accompanied by the generation of Δψ, i.e., the formation of the diffusion potential. It is assumed that along with induction of calcium efflux from the matrix, bile acids are also capable of transporting hydrogen and potassium ions in the opposite direction, i.e., perform H⁺/Ca²⁺ and K⁺/Ca²⁺ exchange. It was found that ruthenium red added to Ca²⁺-loaded energized mitochondria prevents the return of these ions to the matrix and weakens the effect of chenodeoxycholic acid as an inducer of the CsA-sensitive mitochondrial pore and the effect of ursodeoxycholic acid as an inducer of CsA-insensitive permeability of the inner mitochondrial membrane. We conclude that in the conditions of the calcium uniporter activity decrease, Ca²⁺ efflux from the matrix induced by bile acids can be considered as one of the mechanisms reducing their effectiveness as inducers of the Ca²⁺-dependent CsA-sensitive pore and CsA-insensitive permeability transition in mitochondria.     

Mitochondrial Ca<Superscript>2+</Superscript> and regulation of the permeability transition pore

The mitochondrial permeability transition pore was originally described in the 1970’s as a Ca²⁺ activated pore and has since been attributed to the pathogenesis of many diseases. Here we evaluate how each of the current models of the pore complex fit to what is known about how Ca²⁺ regulates the pore, and any insight that provides into the molecular identity of the pore complex. We also discuss the central role of Ca²⁺ in modulating the pore’s open probability by directly regulating processes, such as ATP/ADP balance through the tricarboxylic acid cycle, electron transport chain, and mitochondrial membrane potential. We review how Ca²⁺ influences second messengers such as reactive oxygen/nitrogen species production and polyphosphate formation. We discuss the evidence for how Ca²⁺ regulates post-translational modification of cyclophilin D including phosphorylation by glycogen synthase kinase 3 beta, deacetylation by sirtuins, and oxidation/ nitrosylation of key residues. Lastly we introduce a novel view into how Ca²⁺ activated proteolysis through calpains in the mitochondria may be a driver of sustained pore opening during pathologies such as ischemia reperfusion injury.     

Diacylglycerols Activate Mitochondrial Cationic Channel(s) and Release Sequestered Ca<Superscript>2+</Superscript>

Mitochondria contribute to cytosolic Ca²⁺ homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAGs) induce Ca²⁺ release from Ca²⁺-loaded mammalian mitochondria. Release is not mediated by the uniporter or the Na⁺/Ca²⁺ exchanger, nor is it attributed to putative catabolites. DAGs-induced Ca²⁺ efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca²⁺ and relatively slow reuptake, a secondary progressive release of Ca²⁺ occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAGs-induced Ca²⁺ efflux is abolished by La³⁺ (1 mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca²⁺ efflux. Ca²⁺-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAGs-induced Ca²⁺ release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brain mitoplasts reveal DAGs-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La³⁺. The presence of a second messenger-sensitive Ca²⁺ release mechanism in mitochondria could have an important impact on intracellular Ca²⁺ homeostasis.     

A comparative study of the effects of Pr<Superscript>3+</Superscript> and La<Superscript>3+</Superscript> ions on calcium dependent processes in frog cardiac muscle and rat heart mitochondria

The inotropic effect of Pr³⁺ and La³⁺ ions on the heart muscle of frog Rana ridibunda, as well as the influence of the ions on respiration, swelling, and the potential (ΔΨ_mito) on the inner membrane of Ca²⁺- loaded rat heart mitochondria, energized by glutamate and malate or succinate in the presence of rotenone were studied. It was found that 2 mM Pr³⁺ in Ringer’s solution reduces the force of spontaneous contractions and those induced by electrical stimulation in the heart; it had a negative chronotropic effect, decreasing the frequency of spontaneous contractions. Pr³⁺ and La³⁺ prevented a decrease in the 2,4-dinitrophenol (DNP)- uncoupled respiration of energized rat heart mitochondria, swelling of these organelles in salt media, and a reduction in ΔΨ_mito on the inner mitochondrial membrane that were induced by Ca²⁺ ions. Retardation by Pr³⁺ and La³⁺ ions of these calcium-induced effects may suggest that in the inner mitochondrial membrane these metals inhibit the opening of the mitochondrial permeability transition pore caused by Ca²⁺ overload of mitochondria. The data we obtained are important for a better understanding of the mechanisms of the damaging action of rare-earth elements on Ca²⁺-dependent processes in the vertebrate myocardium.     

Ursodeoxycholic acid,in contrast to other bile acids,induces Ca<Superscript>2+</Superscript>-dependent cyclosporin A-insensitive permeability transition in liver mitochondria in the presence of potassium chloride

The effects of hydrophobic and hydrophilic bile acids as inducers of Ca²⁺-dependent permeability of the inner membrane were studied on isolated liver mitochondria. It is shown that in the absence of the inorganic phosphate (P_i)–a modulator of the mitochondrial pore–hydrophobic bile acids (lithocholic, deoxycholic, chenodeoxycholic) at concentrations of 20–50 μM, as well as a hydrophilic cholic acid at a concentration of 800 μM, induce swelling of liver mitochondria loaded with Ca²⁺. This effect is completely eliminated by a specific inhibitor of mitochondrial pore cyclosporin A (CsA). The effect of the bile acids as inducers of Ca²⁺-dependent CsA-sensitive mitochondrial pore is not associated with the modulation of the P_i effects. In contrast to other tested bile acids, a hydrophilic ursodeoxycholic acid (UDCA) at a concentration of 400 μM is able to induce Ca²⁺-dependent CsA-sensitive pore opening in liver mitochondria only in the presence of P_i or in the absence of potassium chloride in the incubation medium. In the presence of potassium chloride but in the absence of P_i, UDCA effects associated with the induction of the inner membrane permeability (swelling of mitochondria, drop in Δψ, and Ca²⁺ release from the matrix) are also observed in the presence of CsA. This Ca²⁺-dependent permeability of the inner membrane, in contrast to the “classical” CsA-sensitive pore, is characterized by a lower intensity of the mitochondrial swelling, a total drop in Δψ, and Ca²⁺ release from the matrix and is blocked by P_i. We suggest that the induction of the CsA-insensitive permeability in the inner mitochondrial membrane by UDCA is associated with activation of electrophoretic influx of K⁺ into the matrix and Ca²⁺ release from the matrix in exchange to H+. The effect of P_i as a blocker of such permeability is discussed.     

Intracellular Ca<Superscript>2+</Superscript> Pools and Fluxes in Cardiac Muscle-Derived H9c2 Cells

Relevant Ca²⁺ pools and fluxes in H9c2 cells have been studied using fluorescent indicators and Ca²⁺-mobilizing agents. Vasopressin produced a cytoplasmic Ca²⁺ peak with half-maximal effective concentration of 6 nM, whereas thapsigargin-induced Ca²⁺ increase showed half-maximal effect at 3 nM. Depolarization of the mitochondrial inner membrane by protonophore was also associated with an increase in cytoplasmic Ca²⁺. Ionomycin induced a small and sustained depolarization, while thapsigargin had a small but transient effect. The thapsigargin-sensitive Ca²⁺ pool was also sensitive to ionomycin, whereas the protonophore-sensitive Ca²⁺ pool was not. The vasopressin-induced cytoplasmic Ca²⁺ signal, which caused a reversible discharge of the sarco-endoplasmic reticulum Ca²⁺ pool, was sensed as a mitochondrial Ca²⁺ peak but was unaffected by the permeability transition pore inhibitor cyclosporin A. The mitochondrial Ca²⁺ peak was affected by cyclosporin A when the Ca²⁺ signal was induced by irreversible discharge of the intracellular Ca²⁺ pool, i.e., adding thapsigargin. These observations indicate that the mitochondria interpret the cytoplasmic Ca²⁺ signals generated in the reticular store.     

Mitochondrial Ca<Superscript>2+</Superscript> uptake pathways

Calcium (Ca²⁺) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca²⁺ is crucial for the control of energy production and cellular responses to metabolic stress. Ca²⁺ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca²⁺ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca²⁺ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca²⁺ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca²⁺. In view of the fundamental importance of Ca²⁺ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca²⁺ entry. We review the experimental evidence for the existence of alternative Ca²⁺ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca²⁺ signalling.     

Ca<Superscript>2+</Superscript> channels and Ca<Superscript>2+</Superscript> signals involved in abiotic stress responses in plant cells: recent advances

Calcium (Ca²⁺) signals are essential transducers and regulators in many adaptive and developmental processes in plants. Protective responses of plants to a variety of environmental stress factors are mediated by transient changes of Ca²⁺ concentration in plant cells. Ca²⁺ ions are quickly transported by channel proteins present on the plasma membrane. During responses to external stimuli, various signal molecules are transported directly from extracellular to intracellular compartments via Ca²⁺ channel proteins. Three types of Ca²⁺ channels have been identified in plant cell membranes: voltage-dependent Ca²⁺-permeable channels (VDCCs), which is sorted to depolarization-activated Ca²⁺-permeable channels (DACCs) and hyperpolarization-activated Ca²⁺-permeable channels (HACCs), voltage-independent Ca²⁺-permeable channels (VICCs). They make functions in the abiotic stress such as TPCs, CNGCs, MS channels, annexins which distribute in the organelles, plasma membrane, mitochondria, cytosol, intracelluar membrane. This review summarizes recent advances in our knowledge of many types of Ca²⁺ channels and Ca²⁺ signals involved in abiotic stress resistance and responses in plant cells.     

Life after the birth of the mitochondrial Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger,NCLX

Powered by the mitochondrial membrane potential, Ca²⁺ permeates the mitochondria via a Ca²⁺ channel termed Ca²⁺ uniporter and is pumped out by a Na⁺/Ca²⁺ exchanger, both of which are located on the inner mitochondrial membrane. Mitochondrial Ca²⁺ transients are critical for metabolic activity and regulating global Ca²⁺ responses. On the other hand, failure to control mitochondrial Ca²⁺ is a hallmark of ischemic and neurodegenerative diseases. Despite their importance, identifying the uniporter and exchanger remains elusive and their inhibitors are non-specific. This review will focus on the mitochondrial exchanger, initially describing how it was molecularly identified and linked to a novel member of the Na⁺/Ca²⁺ exchanger superfamily termed NCLX. Molecular control of NCLX expression provides a selective tool to determine its physiological role in a variety of cell types. In lymphocytes, NCLX is essential for refilling the endoplasmic reticulum Ca²⁺ stores required for antigendependent signaling. Communication of NCLX with the store-operated channel in astroglia controls Ca²⁺ influx and thereby neuro-transmitter release and cell proliferation. The refilling of the Ca²⁺ stores in the sarcoplasmic reticulum, which is controlled by NCLX, determines the frequency of action potential and Ca²⁺ transients in cardiomyocytes. NCLX is emerging as a hub for integrating glucose-dependent Na⁺ and Ca²⁺ signaling in pancreatic β cells, and the specific molecular control of NCLX expression resolved the controversy regarding its role in neurons and β cells. Future studies on an NCLX knockdown mouse model and identification of human NCLX mutations are expected to determine the role of mitochondrial Ca²⁺ efflux in organ activity and whether NCLX inactivation is linked to ischemic and/or neurodegenerative syndromes. Structure-function analysis and protein analysis will identify the NCLX mode of regulation and its partners in the inner membrane of the mitochondria.     

Cooperative Interactions in Energy-dependent Accumulation of Ca<Superscript>2+</Superscript> by Isolated Rat Liver Mitochondria

THE energy-dependent accumulation of Ca²⁺ by isolated rat liver mitochondria is intimately associated with oxidative phosphorylation¹. Available evidence supports the idea that, like the permeases postulated for some mitochondrial metabolites², this active accumulation of Ca²⁺ may involve a “carrier” in the mitochondrial membrane specific for Ca²⁺ (ref. 3). Several studies have shown that the energy-independent “binding” of Ca²⁺ to sites on the (inner membrane of), intact mitochondria and of submitochondrial particles exhibits hyperbolic saturation curves as a function of Ca²⁺ concentration^{4, 5}.     

Mitochondrial Ca<Superscript>2+</Superscript> cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca²⁺ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca²⁺, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca²⁺ and the swelling of mitochondria. Inhibitors of the Ca²⁺ uniporter, ruthenium red and La³⁺, as well as EGTA added in 10 min after the Pal/Ca²⁺-activated pore opening, prevent the release of Ca²⁺ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr²⁺], which leads to the activation of phospholipase A₂ and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca²⁺ cycle, in which Ca²⁺ uptake is mediated by the Ca²⁺ uniporter and Ca²⁺ efflux occurs via a short-living Pal/Ca²⁺-activated pore.     

Distinctive characteristics and functions of multiple mitochondrial Ca<Superscript>2+</Superscript> influx mechanisms

Intracellular Ca²⁺ is vital for cell physiology. Disruption of Ca²⁺ homeostasis contributes to human diseases such as heart failure, neuron-degeneration, and diabetes. To ensure an effective intracellular Ca²⁺ dynamics, various Ca²⁺ transport proteins localized in different cellular regions have to work in coordination. The central role of mitochondrial Ca²⁺ transport mechanisms in responding to physiological Ca²⁺ pulses in cytosol is to take up Ca²⁺ for regulating energy production and shaping the amplitude and duration of Ca²⁺ transients in various micro-domains. Since the discovery that isolated mitochondria can take up large quantities of Ca²⁺ approximately 5 decades ago, extensive studies have been focused on the functional characterization and implication of ion channels that dictate Ca²⁺ transport across the inner mitochondrial membrane. The mitochondrial Ca²⁺ uptake sensitive to non-specific inhibitors ruthenium red and Ru360 has long been considered as the activity of mitochondrial Ca²⁺ uniporter (MCU). The general consensus is that MCU is dominantly or exclusively responsible for the mitochondrial Ca²⁺ influx. Since multiple Ca²⁺ influx mechanisms (e.g. L-, T-, and N-type Ca²⁺ channel) have their unique functions in the plasma membrane, it is plausible that mitochondrial inner membrane has more than just MCU to decode complex intracellular Ca²⁺ signaling in various cell types. During the last decade, four molecular identities related to mitochondrial Ca²⁺ influx mechanisms have been identified. These are mitochondrial ryanodine receptor, mitochondrial uncoupling proteins, LETM1 (Ca²⁺/H⁺ exchanger), and MCU and its Ca²⁺ sensing regulatory subunit MICU1. Here, we briefly review recent progress in these and other reported mitochondrial Ca²⁺ influx pathways and their differences in kinetics, Ca²⁺ dependence, and pharmacological characteristics. Their potential physiological and pathological implications are also discussed.     

Ca<Superscript>2+</Superscript>-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca<Superscript>2+</Superscript> Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca²⁺ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca²⁺. We show that hyperphosphorylation by exogenous PKA increases open probability (P _o) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca²⁺; lowering [Ca²⁺] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca²⁺-sensitive mode of gating. We additionally found that cytosolic incubation with Mg²⁺/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca²⁺. We show that the endogenous kinase is both Ca²⁺-dependent and sensitive to inhibitors of PKC. Moreover, the Ca²⁺-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca²⁺-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca²⁺ regulation.     

Disruption of actin filaments induces mitochondrial Ca<Superscript>2+</Superscript> release to the cytoplasm and [Ca<Superscript>2+</Superscript>]<Subscript>c</Subscript> changes in <Emphasis Type="Italic">Arabidopsis</Emphasis>root hairs

Background  

Mitochondria are dynamic organelles that move along actin filaments, and serve as calcium stores in plant cells. The positioning and dynamics of mitochondria depend on membrane-cytoskeleton interactions, but it is not clear whether microfilament cytoskeleton has a direct effect on mitochondrial function and Ca²⁺ storage. Therefore, we designed a series of experiments to clarify the effects of actin filaments on mitochondrial Ca²⁺ storage, cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c), and the interaction between mitochondrial Ca²⁺ and cytoplasmic Ca²⁺ in Arabidopsis root hairs.     

The role of mitochondrial palmitate/Ca<Superscript>2+</Superscript>-activated pore in palmitate-induced apoptosis

The evidence of possible involvement of the mitochondrial cyclosporin A-insensitive palmitate/Ca²⁺-activated pore in palmitate-induced apoptosis is presented. It has been established that the opening of the palmitate/Ca²⁺-activated pore results in the high-amplitude swelling of mitochondria and the release of the apoptosis-inducing factor from organelles. These processes are accompanied by a short-term slight decrease of membrane potential, which recovers in 1 min. The possible role of the palmitate/Ca²⁺-activated pore in the induction of palmitate-induced apoptosis is discussed.     

Effect of Mg<Superscript>2+</Superscript> versus Ca<Superscript>2+</Superscript> on the behavior of Annexin A5 in a membrane-bound state

Annexin A5 (AnxA5) binds to negatively charged phospholipid membranes in a Ca²⁺ dependent manner. Several studies already demonstrate that Mg²⁺ ions cannot induce the binding. In this paper, quartz crystal microbalance with dissipation monitoring (QCM-D), Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) and molecular dynamics (MD) were performed to elucidate the high specificity of Ca²⁺ versus Mg²⁺ on AnxA5 binding to membrane models. In the presence of Ca²⁺, AnxA5 showed a strong interaction with lipids, the protein is adsorbed mainly in α-helix under the DMPS monolayer, with an orientation of the α-helices axes slightly tilted with respect to the normal of the phospholipid monolayer as revealed by PMIRRAS. The Ca²⁺ ions interact strongly with the phosphate group of the phospholipid monolayer. In the presence of Mg²⁺, instead of Ca²⁺, no interaction of AnxA5 with lipids was detected. Molecular dynamics simulations allow us to explain the high specificity of calcium. Ca²⁺ ions are well exposed and surrounded by labile water molecules at the surface of the protein, which then favour their binding to the phosphate group of the membrane, explaining their specificity. To the contrary, Mg²⁺ ions are embedded in the protein structure, with a smaller number of water molecules strongly bound. We conclude that the embedded Mg²⁺ ions inside the AnxA5 structure are not able to link the protein to the phosphate group of the phospholipids for this reason.     

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.     

Ca<Superscript>2+</Superscript>-ATPase in the symbiosome membrane from broad bean root nodules: further evidence for its functioning as ATP-driven Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchanger

To date, it has been established that the symbiosome membrane (SM), i.e., plant-derived membrane of symbiosomes, nitrogen-fixing compartments of legume root nodules, is equipped with Ca²⁺-ATPase transporting Ca²⁺ ions through the SM from the cytosol of infected cells into the symbiosome space (SS). Earlier in the experiments on the SM vesicles isolated from broad bean root nodules some data indicating the action of the Ca²⁺-ATPase as ATP-driven Ca²⁺/H⁺ antiporter were obtained. In the present work performed on isolated symbiosomes from the same plant object, further evidence in favor of calcium-proton countertransport mechanism of the pump operation was obtained. These were expressed in vanadate-sensitive alkalinization of the SS coupled with Ca²⁺ uptake by symbiosomes catalyzed by the SM Ca²⁺-ATPase, stimulation of the kinetics of the latter process in the response to artificial acidification of the SS and expectable modulation of ITP-hydrolyzing activity of this enzyme caused by the variation of pH within this compartment. The above findings are discussed in the framework of the model describing the mechanism of Ca²⁺-ATPase operation as an ATP-driven Ca²⁺/H⁺ exchanger and on this base allow us to put forward the hypothesis about the involvement of this enzyme in symbiosome signaling in a Ca²⁺- and pH-dependent manner.     

Intracellular Free Ca<Superscript>2+</Superscript> and Membrane Properties of Motoneurones

IN some recent experiments on cats^1,2, it was found that 2,4-dinitrophenol strongly depresses the excitability of cortical neurones, probably by causing an increase in membrane conductance to K⁺ (g_K). It was suggested that the immediate cause of this effect may be an increased amount of free Ca²⁺ inside the neurone, resulting from slowing down of Ca²⁺ uptake by mitochondria (compare ref. 3). Although this idea is supported by the finding⁴ that injections of Ca²⁺ into neurones in Aplysia greatly increase membrane g_K, no comparable experiments have been performed in a more closely related species. The present communication describes some effects produced by injections of Ca²⁺ into mammalian neurones.     

Ca<Superscript>2+</Superscript> Buffering Capacity of Mitochondria After Oxygen-Glucose Deprivation in Hippocampal Neurons

In an earlier study, we showed that mitochondria hyperpolarized after short periods of oxygen-glucose deprivation (OGD), and this response appeared to be associated with subsequent apoptosis or survival. Here, we demonstrated that hyperpolarization following short periods of OGD (30 min; 30OGD group) increased the cytosolic Ca²⁺ ([Ca²⁺]_c) buffering capacity in mitochondria. After graded OGD (0 min (control), 30 min, 120 min), rat cultured hippocampal neurons were exposed to glutamate, evoking Ca²⁺influx. The [Ca²⁺]_c level increased sharply, followed by a rapid increase in mitochondrial Ca²⁺ [Ca²⁺]_m. The increase in the [Ca²⁺]_m level accompanied a reduction in the [Ca²⁺]_c level. After reaching a peak, the [Ca²⁺]_c level decreased more rapidly in the 30OGD group than in the control group. This buffering reaction was pronounced in the 30OGD group, but not in the 120OGD group. The enhanced buffering capacity of the mitochondria may be linked to preconditioning after short-term ischemic episodes.