Extramitochondrial Ca2+ in the Nanomolar Range Regulates Glutamate-Dependent Oxidative Phosphorylation on Demand

We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca²⁺ in physiological concentration ranges (S_0.5 = 360 nM Ca²⁺). This regulation was not affected by RR, an inhibitor of the mitochondrial Ca²⁺ uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca²⁺-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca²⁺. At micromolar concentrations, Ca²⁺ can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca²⁺ regulation of respiration/OXPHOS via aralar. These results imply a new mode of glutamate-dependent OXPHOS regulation as a demand-driven regulation of mitochondrial function. This regulation involves the mitochondrial glutamate/aspartate carrier aralar which controls mitochondrial substrate supply according to the level of extramitochondrial Ca²⁺.     

The regulation of OXPHOS by extramitochondrial calcium

Despite extensive research, the regulation of mitochondrial function is still not understood completely. Ample evidence shows that cytosolic Ca²⁺ has a strategic task in co-ordinating the cellular work load and the regeneration of ATP by mitochondria. Currently, the paradigmatic view is that Ca_cyt²⁺ taken up by the Ca²⁺ uniporter activates the matrix enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and isocitrate dehydrogenase. However, we have recently found that Ca²⁺ regulates the glutamate-dependent state 3 respiration by the supply of glutamate to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier. Since this activation is not affected by ruthenium red, glutamate transport into mitochondria is controlled exclusively by extramitochondrial Ca²⁺. Therefore, this discovery shows that besides intramitochondrial also extramitochondrial Ca²⁺ regulates oxidative phosphorylation. This new mechanism acts as a mitochondrial “gas pedal”, supplying the OXPHOS with substrate on demand. These results are in line with recent findings of Satrustegui and Palmieri showing that aralar as part of the malate–aspartate shuttle is involved in the Ca²⁺-dependent transport of reducing hydrogen equivalents (from NADH) into mitochondria. This review summarises results and evidence as well as hypothetical interpretations of data supporting the view that at the surface of mitochondria different regulatory Ca²⁺-binding sites exist and can contribute to cellular energy homeostasis. Moreover, on the basis of our own data, we propose that these surface Ca²⁺-binding sites may act as targets for neurotoxic proteins such as mutated huntingtin and others. The binding of these proteins to Ca²⁺-binding sites can impair the regulation by Ca²⁺, causing energetic depression and neurodegeneration.     

Neurons Efficiently Repair Glutamate-induced Oxidative DNA Damage by a Process Involving CREB-mediated Up-regulation of Apurinic Endonuclease 1

Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca²⁺ influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca²⁺ chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knockdown induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca²⁺/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca²⁺- and mitochondrial reactive oxygen species-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca²⁺-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors.     

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.     

The Mitochondrial Aspartate/Glutamate Carrier AGC1 and Calcium Homeostasis: Physiological Links and Abnormalities in Autism

Autism spectrum disorder (ASD) is a severe, complex neurodevelopmental disorder characterized by impairments in reciprocal social interaction and communication, and restricted and stereotyped patterns of interests and behaviors. Recent evidence has unveiled an important role for calcium (Ca²⁺) signaling in the pathogenesis of ASD. Post-mortem studies of autistic brains have pointed toward abnormalities in mitochondrial function as possible downstream consequences of altered Ca²⁺ signaling, abnormal synapse formation, and dysreactive immunity. SLC25A12, an ASD susceptibility gene, encodes the Ca²⁺-regulated mitochondrial aspartate–glutamate carrier, isoform 1 (AGC1). AGC1 is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and adenosine triphosphate (ATP) production. Here, we review the physiological roles of AGC1, its links to calcium homeostasis, and its involvement in autism pathogenesis.     

l-Aspartate but Not the d Form Is Secreted Through Microvesicle-Mediated Exocytosis and Is Sequestered Through Na+-Dependent Transporter in Rat Pinealocytes

Abstract: Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca²⁺ ionophore) in the presence of Ca²⁺ and was proportional to the amount of l -glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L- or N-type voltage-gated Ca²⁺ channel blockers. Bay K 8644, an agonist for the L-type Ca²⁺ channel, stimulated the release of aspartate 2.1-fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the l form, although pinealocytes also contain the d form in their cytoplasm, corresponding to ～30% of the total free aspartate. Radiolabeled l -aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na⁺-dependent manner. The Na⁺-dependent uptake of l -aspartate was strongly inhibited by l -cysteine sulfinate, β-hydroxyaspartate, and l -serine-O-sulfate, inhibitors for the Na⁺-dependent glutamate/aspartate transporter on the plasma membrane. Na⁺-dependent sequestration of l -aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that l -aspartate is released through microvesicle-mediated exocytosis from pinealocytes and is taken up again through the Na⁺-dependent transporter at the plasma membrane. The possible role of l -aspartate as an intercellular chemical transmitter in the pineal gland is discussed.     

Calcium Sequestering Ability of Mitochondria Modulates Influx of Calcium through Glutamate Receptor Channel

The excitotoxicity of glutamate is believed to be mediated by sustained increase in the cytosolic Ca²⁺ concentration. Mitochondria play a vital role in buffering the cytosolic calcium overload in stimulated neurons. Here we have studied the glutamate induced Ca²⁺ signals in cortical brain slices under physiological conditions and the conditions that modify the mitochondrial functions. Exposure of slices to glutamate caused a rapid increase in [Ca²⁺]_i followed by a slow and persistently rising phase. The rapid increase in [Ca²⁺]_i was mainly due to influx of Ca²⁺ through the N-methyl-D-aspartate (NMDA) receptor channels. Glutamate stimulation in the absence of Ca²⁺ in the extracellular medium elicited a small transient rise in [Ca²⁺]_i which can be attributed to the mobilization of Ca²⁺ from IP₃ sensitive endoplasmic reticulum pools consequent to activation of metabotropic glutamate receptors. The glutamate induced Ca²⁺ influx was accompanied by depolarization of the mitochondrial membrane, which was inhibited by ruthenium red, the blocker of mitochondrial Ca²⁺ uniporter. These results imply that mitochondria sequester the Ca²⁺ loaded into the cytosol by glutamate stimulation. Persistent depolarization of mitochondrial membrane observed in presence of extracellular Ca²⁺ caused permeability transition and released the sequestered Ca²⁺ which is manifested as slow rise in [Ca²⁺]_i. Protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) depolarized the mitochondrial membrane and enhanced the glutamate induced [Ca²⁺]_i response. Contrary to this, treatment of slices with mitochondrial inhibitor oligomycin or ruthenium red markedly reduced the [Ca²⁺]_i response. Combined treatment with oligomycin and rotenone further diminished the [Ca²⁺]_i response and also abolished the CCCP mediated rise in [Ca²⁺]_i. However, rotenone alone had no effect on glutamate induced [Ca²⁺]_i response. These changes in glutamate-induced [Ca²⁺]_i response could not be explained on the basis of deficient mitochondrial Ca²⁺ sequestration or ATP dependent Ca²⁺ buffering. The mitochondrial inhibitors reduced the cellular ATP/ADP ratio, however, this would have restrained the ATP dependent Ca²⁺ buffering processes leading to elevation of [Ca²⁺]_i. In contrast our results showed repression of Ca²⁺ signal except in case of CCCP which drastically reduced the ATP/ADP ratio. It was inferred that, under the conditions that hamper the Ca²⁺ sequestering ability of mitochondria, the glutamate induced Ca²⁺ influx could be impeded. To validate this, influx of Mn²⁺ through ionotropic glutamate receptor channel was monitored by measuring the quenching of Fura-2 fluorescence. Treatment of slices with oligomycin and rotenone prior to glutamate exposure conspicuously reduced the rate of glutamate induced fluorescence quenching as compared to untreated slices. Thus our data establish that the functional status of mitochondria can modify the activity of ionotropic glutamate receptor and suggest that blockade of mitochondrial Ca²⁺ sequestration may desensitize the NMDA receptor operated channel.     

Regulation of neuronal energy metabolism by calcium: Role of MCU and Aralar/malate-aspartate shuttle

Calcium is a major regulator of cellular metabolism. Calcium controls mitochondrial respiration, and calcium signaling is used to meet cellular energetic demands through energy production in the organelle. Although it has been widely assumed that Ca²⁺-actions require its uptake by mitochondrial calcium uniporter (MCU), alternative pathways modulated by cytosolic Ca²⁺ have been recently proposed. Recent findings have indicated a role for cytosolic Ca²⁺ signals acting on mitochondrial NADH shuttles in the control of cellular metabolism in neurons using glucose as fuel. It has been demonstrated that AGC1/Aralar, the component of the malate/aspartate shuttle (MAS) regulated by cytosolic Ca²⁺, participates in the maintenance of basal respiration exerted through Ca²⁺-fluxes between ER and mitochondria, whereas mitochondrial Ca²⁺-uptake by MCU does not contribute. Aralar/MAS pathway, activated by small cytosolic Ca²⁺ signals, provides in fact substrates, redox equivalents and pyruvate, fueling respiration. Upon activation and increases in workload, neurons upregulate OxPhos, cytosolic pyruvate production and glycolysis, together with glucose uptake, in a Ca²⁺-dependent way, and part of this upregulation is via Ca²⁺ signaling. Both MCU and Aralar/MAS contribute to OxPhos upregulation, Aralar/MAS playing a major role, especially at small and submaximal workloads. Ca²⁺ activation of Aralar/MAS, by increasing cytosolic NAD⁺/NADH provides Ca²⁺-dependent increases in glycolysis and cytosolic pyruvate production priming respiration as a feed-forward mechanism in response to workload. Thus, except for glucose uptake, these processes are dependent on Aralar/MAS, whereas MCU is the relevant target for Ca²⁺ signaling when MAS is bypassed, by using pyruvate or β-hydroxybutyrate as substrates.     

Uncoupling Protein 2 (UCP2) Function in the Brain as Revealed by the Cerebral Metabolism of (1–<Superscript>13</Superscript>C)-Glucose

The mitochondrial aspartate/glutamate transporter Aralar/AGC1/Slc25a12 is critically involved in brain aspartate synthesis, and AGC1 deficiency results in a drastic fall of brain aspartate levels in humans and mice. It has recently been described that the uncoupling protein UCP2 transports four carbon metabolites including aspartate. Since UCP2 is expressed in several brain cell types and AGC1 is mainly neuronal, we set to test whether UCP2 could be a mitochondrial aspartate carrier in the brain glial compartment. The study of the cerebral metabolism of (1–¹³C)-glucose in vivo in wild type and UCP2-knockout mice showed no differences in C3 or C2 labeling of aspartate, suggesting that UCP2 does not function as a mitochondrial aspartate carrier in brain. However, surprisingly, a clear decrease (of about 30–35?%) in the fractional enrichment of glutamate, glutamine and GABA was observed in the brains of UCP2-KO mice which was not associated with differences in either glucose or lactate enrichments. The results suggest that the dilution in the labeling of glutamate and its downstream metabolites could originate from the uptake of an unlabeled substrate that could not leave the matrix via UCP2 becoming trapped in the matrix. Understanding the nature of the unlabeled substrate and its precursor(s) as alternative substrates to glucose is of interest in the context of neurological diseases associated with UCP2.     

Cytosolic and mitochondrial Ca2+ concentrations in primary hepatocytes change with ageing and in consequence of an mtDNA mutation

Mitochondrial Ca²⁺ flux is crucial for the regulation of cell metabolism. Ca²⁺ entry to the mitochondrial matrix is mediated by VDAC1 and MCU with its regulatory molecules. We investigated hepatocytes isolated from conplastic C57BL/6NTac-mt^NODLtJ mice (mtNOD) that differ from C57BL/6NTac mice (controls) by a point mutation in mitochondrial-encoded subunit 3 of cytochrome c oxidase, resulting in functional and morphological mitochondrial adaptations. Mice of both strains up to 12 months old were compared using mitochondrial GEM-GECO1 and cytosolic CAR-GECO1 expression to gain knowledge of age-dependent alterations of Ca²⁺ concentrations. In controls we observed a significant increase in glucose-induced cytosolic Ca²⁺ concentration with ageing, but only a minor elevation in mitochondrial Ca²⁺ concentration. Conversely, glucose-induced mitochondrial Ca²⁺ concentration significantly declined with ageing in mtNOD mice, paralleled by a slight decrease in cytosolic Ca²⁺ concentration. This was consistent with a significant reduction of the MICU1 to MCU expression ratio and a decline in MCUR1. Our results can best be explained in terms of the adaptation of Ca²⁺ concentrations to the mitochondrial network structure. In the fragmented mitochondrial network of ageing controls there is a need for high cytosolic Ca²⁺ influx, because only some of the isolated mitochondria are in direct contact with the endoplasmic reticulum. This is not important in the hyper-fused elongated mitochondrial network found in ageing mtNOD mice which facilitates rapid Ca²⁺ distribution over a large mitochondrial area.     

Sustained intracellular calcium rise mediates neuronal mitophagy in models of autosomal dominant optic atrophy

Mitochondrial dysfunction and mitophagy are often hallmarks of neurodegenerative diseases such as autosomal dominant optic atrophy (ADOA) caused by mutations in the key mitochondrial dynamics protein optic atrophy 1 (Opa1). However, the second messengers linking mitochondrial dysfunction to initiation of mitophagy remain poorly characterized. Here, we show in mammalian and nematode neurons that Opa1 mutations trigger Ca²⁺-dependent mitophagy. Deletion or expression of mutated Opa1 in mouse retinal ganglion cells and Caenorhabditis elegans motor neurons lead to mitochondrial dysfunction, increased cytosolic Ca²⁺ levels, and decreased axonal mitochondrial density. Chelation of Ca²⁺ restores mitochondrial density in neuronal processes, neuronal function, and viability. Mechanistically, sustained Ca²⁺ levels activate calcineurin and AMPK, placed in the same genetic pathway regulating axonal mitochondrial density. Our data reveal that mitophagy in ADOA depends on Ca²⁺-calcineurin-AMPK signaling cascade.Subject terms: Cell biology, Neurological disorders     

Osthole and imperatorin, the active constituents of Cnidium monnieri (L.) Cusson, facilitate glutamate release from rat hippocampal nerve terminals

We examined the effects of osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, on the release of glutamate from rat hippocampal synaptosomes and investigated the possible mechanism. The results showed that osthole or imperatorin significantly facilitated 4-aminopridine (4-AP)-evoked glutamate release in a concentration-dependent manner. The facilitatory action of osthole or imperatorin was blocked by the vesicular transporter inhibitor bafilomycin A1, not by the glutamate transporter inhibitor l-transpyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), indicating that the release facilitation by osthole or imperatorin results from a enhancement of vesicular exocytosis and not from an increase of Ca²⁺-independent efflux via glutamate transporter. Examination of the effect of osthole and imperatorin on cytosolic [Ca²⁺] revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca²⁺ influx. Consistent with this, ω-conotoxin MVIIC, a wide-spectrum blocker of the N- and P/Q-type Ca²⁺ channels, significantly suppressed the osthole or imperatorin-mediated facilitation of glutamate release, but intracellular Ca²⁺ release inhibitor dantrolene had no effect. Osthole or imperatorin did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization; thus, the facilitation of 4-AP-evoked Ca²⁺ influx and glutamate release produced by osthole or imperatorin was not due to it decreasing synaptosomal excitability. In addition, osthole or imperatorin-mediated inhibition of 4-AP-evoked release was prevented by protein kinase C (PKC) inhibitors. Furthermore, osthole or imperatorin increased 4-AP-induced phosphorylation of PKC. Together, these results suggest that osthole or imperatorin effects a facilitation of glutamate release from nerve terminals by positively modulating N-and P/Q-type Ca²⁺ channel activation through a signaling cascade involving PKC.     

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.     

Osthole and imperatorin,the active constituents of Cnidium monnieri (L.) Cusson,facilitate glutamate release from rat hippocampal nerve terminals

We examined the effects of osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, on the release of glutamate from rat hippocampal synaptosomes and investigated the possible mechanism. The results showed that osthole or imperatorin significantly facilitated 4-aminopridine (4-AP)-evoked glutamate release in a concentration-dependent manner. The facilitatory action of osthole or imperatorin was blocked by the vesicular transporter inhibitor bafilomycin A1, not by the glutamate transporter inhibitor l-transpyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), indicating that the release facilitation by osthole or imperatorin results from a enhancement of vesicular exocytosis and not from an increase of Ca²⁺-independent efflux via glutamate transporter. Examination of the effect of osthole and imperatorin on cytosolic [Ca²⁺] revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca²⁺ influx. Consistent with this, ω-conotoxin MVIIC, a wide-spectrum blocker of the N- and P/Q-type Ca²⁺ channels, significantly suppressed the osthole or imperatorin-mediated facilitation of glutamate release, but intracellular Ca²⁺ release inhibitor dantrolene had no effect. Osthole or imperatorin did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization; thus, the facilitation of 4-AP-evoked Ca²⁺ influx and glutamate release produced by osthole or imperatorin was not due to it decreasing synaptosomal excitability. In addition, osthole or imperatorin-mediated inhibition of 4-AP-evoked release was prevented by protein kinase C (PKC) inhibitors. Furthermore, osthole or imperatorin increased 4-AP-induced phosphorylation of PKC. Together, these results suggest that osthole or imperatorin effects a facilitation of glutamate release from nerve terminals by positively modulating N-and P/Q-type Ca²⁺ channel activation through a signaling cascade involving PKC.     

Regulatory sites and effectors ofd-[3H]aspartate release from rat cerebral cortex

To study the effect of agents interfering with the biosynthesis and/or the K⁺-evoked Ca²⁺-dependent release of neurotransmitter glutamate, rat cerebral slices were preincubated with Krebs-Ringer-HEPES-glucose-glutamine buffer (KRH buffer), loaded withd-[³H]aspartate and superfused with the preincubation medium in the presence or in the absence of Ca²⁺. The difference in radioactivity release divided by the basal release per min under the two conditions represented the K⁺-evoked Ca²⁺-dependent release. The agents used were: 1) Aminooxyacetic acid (AOAA), the inhibitor of transaminases, 2) Leucine (Leu), the inhibitor of phosphate activated glutaminase (PAG), 3) NH₄ ⁺, the inhibitor of PAG, 4) Phenylsuccinic acid (Phs), the inhibitor of the mitochondrial ketodicarboxylate carrier, 5) ketone bodies, the inhibitors of glycolysis, 6) the absence of glutamine, the substrate of PAG. The results show that Leu, NH₄ ⁺, Phs and the absence of Gln significantly increase the K⁺-evoked Ca²⁺-dependent release of radioactivity by 64%, 200%, 95% and 147% respectively, indicating that these agents are inhibitors of the K⁺-evoked Ca²⁺-dependent release of glutamate. Ketone bodies and AOAA had no effect. These results indicate that the major if not the exclusive biosynthetic pathway of neurotransmitter glutamate in rat cerebral cortex is through the PAG reaction and support a model for the pathway followed by neurotransmitter glutamate i.e. glutamate formed outside the inner mitochondrial membrane has to enter the mitochondrial matrix or is formed within it from where it can be extruded to supply the transmitter pool in exchange of GABA.     

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na⁺, Ca²⁺) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca²⁺ at the expense of the Na⁺ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca²⁺ extrusion and thus to the recovery from intracellular Ca²⁺ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na⁺ to 1 Ca²⁺, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na⁺ concentration or minor depolarisations switch it into the “reverse mode” (Ca²⁺ import/Na⁺ export). Notably, transient Na⁺ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na⁺-permeable ion channels in response to neuronal activity. Activity-related Na⁺ transients result in NCX reversal, which mediates Ca²⁺ influx from the extracellular space, thereby generating astrocyte Ca²⁺ signalling independent from InsP₃-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca²⁺ overload, while dampening Na⁺ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na⁺-driven, reverse NCX-mediated astrocyte Ca²⁺ signals are involved neurone-glia interaction. Na⁺ transients, translated by the NCX into Ca²⁺ elevations, thereby emerge as a new signalling pathway in astrocytes.     

The Mitochondrial Na+/Ca2+ Exchanger Upregulates Glucose Dependent Ca2+ Signalling Linked to Insulin Secretion

Mitochondria mediate dual metabolic and Ca²⁺ shuttling activities. While the former is required for Ca²⁺ signalling linked to insulin secretion, the role of the latter in β cell function has not been well understood, primarily because the molecular identity of the mitochondrial Ca²⁺ transporters were elusive and the selectivity of their inhibitors was questionable. This study focuses on NCLX, the recently discovered mitochondrial Na⁺/Ca²⁺ exchanger that is linked to Ca²⁺ signalling in MIN6 and primary β cells. Suppression either of NCLX expression, using a siRNA construct (siNCLX) or of its activity, by a dominant negative construct (dnNCLX), enhanced mitochondrial Ca²⁺ influx and blocked efflux induced by glucose or by cell depolarization. In addition, NCLX regulated basal, but not glucose-dependent changes, in metabolic rate, mitochondrial membrane potential and mitochondrial resting Ca²⁺. Importantly, NCLX controlled the rate and amplitude of cytosolic Ca²⁺ changes induced by depolarization or high glucose, indicating that NCLX is a critical and rate limiting component in the cross talk between mitochondrial and plasma membrane Ca²⁺ signalling. Finally, knockdown of NCLX expression was followed by a delay in glucose-dependent insulin secretion. These findings suggest that the mitochondrial Na⁺/Ca²⁺ exchanger, NCLX, shapes glucose-dependent mitochondrial and cytosolic Ca²⁺ signals thereby regulating the temporal pattern of insulin secretion in β cells.     

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca²⁺ influx and mitochondrial Ca²⁺ overload. A substantial change in intracellular Ca²⁺ homeostasis or remodeling of intracellular Ca²⁺ homeostasis may favor neuron damage in old neurons. For investigating Ca²⁺ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca²⁺ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca²⁺ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca²⁺ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca²⁺ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.