Alkalinization Prolongs Recovery from Glutamate-Induced Increases in Intracellular Ca2+ Concentration by Enhancing Ca2+ Efflux Through the Mitochondrial Na+/Ca2+ Exchanger in Cultured Rat Forebrain Neurons

Abstract: Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 µ M , for 15 s)-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in indo-1-loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES-buffered saline solution (HBSS) was 126 ± 30 s, whereas recovery time was 216 ± 19 s when the pH was increased to 8.5. Removal of extracellular Ca²⁺ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca²⁺ recovery by affecting intraneuronal Ca²⁺ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca²⁺ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p -(trifluoromethoxyphenyl)hydrazone (FCCP; 750 n M ). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential-sensitive fluorescent indicator, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1). We found that the effect of pH 8.5 HBSS on Ca²⁺ recovery was completely inhibited by the mitochondrial Na⁺/Ca²⁺ exchange inhibitor CGP-37157 (25 µ M ). This suggests that increased mitochondrial Ca²⁺ efflux via the mitochondrial Na²⁺/Ca²⁺ exchanger is responsible for the prolongation of [Ca²⁺]_i recovery caused by alkaline pH following glutamate exposure.     

A Simple Cranial Window Technique for Optical Monitoring of Cerebrocortical Microcirculation and NAD/NADH Redox State. Effect of Mitochondrial Electron Transport Inhibitors and Anoxic Anoxia

Abstract: Fluorescence of NADH and vascular volume of the brain cortex of chloralose-anesthetized cats were measured by surface fluororeflectometry. A cranial window and superfusion technique was elaborated for the topical inhibition of mitochondrial electron transport in the brain cortex by amytal (inhibits at site I) and cyanide (inhibits at site III). The changes in NAD/NADH redox state and CVV evoked by these electron transport inhibitors were compared with those elicited by anoxic anoxia. Amytal (10^-3-10^-1 M ) and cyanide (10^-5-10^-2 M ) resulted in a concentration-dependent and reversible increase in cortical NAD reduction and vascular volume, but the cerebrocortical vessels were almost completely dilatated long before maximum NAD reduction was reached. Cyanide at 10^-2 M increased cortical NAD reduction and vascular volume as much as anoxic anoxia. Amytal at 10^-1 M induced approximately half of the NAD reduction evoked by 10^-2 M cyanide or anoxic anoxia, but resulted in only slightly less vasodilatation than that following cyanide and anoxic anoxia. Since amytal inhibits mitochondrial electron transport at site I—and cyanide and anoxia at site III—but induces a comparable degree of vasodilatation, it is concluded that cytochrome oxidase cannot be the single molecular oxygen sensor in the brain cortex.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

Nitric Oxide Causes Glutamate Release from Brain Synaptosomes

Abstract: We determined the ability of pathological levels of nitric oxide (NO) to cause glutamate release from isolated rat brain nerve terminals using a fluorometric assay. It was found that NO (0.7 and 2 µ M ) produced (4 and 10 nmol/mg of synaptosomal protein) Ca²⁺-independent glutamate release from synaptosomes (after 1 min of exposure). Spermine/NO complex (spermine NONOate; a slow NO donor) and potassium cyanide (an inhibitor of cytochrome oxidase) also caused Ca²⁺-independent glutamate release. Preincubation of synaptosomes with 5 µ M 1 H -[1,2,4]oxadiazole[4,3- a ]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) had no effect on NO-induced Ca²⁺-independent glutamate release. Ca²⁺-independent glutamate release produced by NO was greater in a low-oxygen medium. NO, spermine NONOate, and potassium cyanide inhibited synaptosomal respiration with a similar order of potency with respect to their ability to cause glutamate release. Because NO has been shown previously to inhibit reversibly cytochrome oxidase in competition with oxygen, our findings in this study suggest that NO (and cyanide) causes glutamate release following inhibition of mitochondrial respiration at the level of cytochrome oxidase. Thus, elevated NO production leading to mitochondrial dysfunction, glutamate release, and excitotoxicity may contribute to neuronal death in neurological diseases.     

Novel Pharmacological Sensitivity of the Presynaptic Calcium Channels Controlling Acetylcholine Release in Skate Electric Organ

Abstract: The presynaptic terminals of skate ( Raja montagui ) electric organ were tested for their sensitivity to calcium channel antagonists. Acetylcholine (ACh) release and the elevation of intraterminal Ca²⁺ concentrations triggered by K⁺ depolarisation were studied. ACh release was measured as ³H efflux from slices of organ prelabelled with [³H]choline. Depolarisation caused a marked, Ca²⁺-dependent increase in ³H efflux that was completely blocked by 100 µ M Cd²⁺ and by 300 n M ω-conotoxin-MVIIC (MVIIC). Inhibition by MVIIC was concentration dependent (IC₅₀ of ∼20 n M ) and reversible. No inhibition was seen with nifedipine (5 µ M ) or the two other peptide antagonists studied: ω-conotoxin-GVIA (GVIA) at 5 µ M and ω-agatoxin-IVA (Aga-IVA) at 1 µ M . In a "nerve plate" preparation (a presynaptic plexus of nerve fibres, Schwann cells, and nerve terminals) changes in intraterminal Ca²⁺ concentrations were measured by microfluorimetry using fluo-3. An increase in fluorescence, indicating a rise in the free [Ca²⁺], rapidly followed K⁺ depolarisation, and this change was restricted to the nerve terminals. This response was insensitive to nifedipine (5 µ M ), GVIA (5 µ M ), and Aga-IVA (300 n M ) but almost completely abolished by MVIIC (1 µ M ). MVIIC inhibition was concentration dependent and partially reversible. These results show that the nerve terminals in skate electric organ have calcium channels with a pharmacological sensitivity that is markedly different from the established L, N, and P types in other systems but shares some, but not all, of the features of the recently described Q type.     

Inhibition of Bradykinin-Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108-15 Cells

Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC₅₀ of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca²⁺]_i increase and had no effect on the EC₅₀ value for bradykinin regardless of the presence of extracellular Ca²⁺. Neither the capacity of ⁴⁵Ca²⁺ accumulation within intracellular nonmitochondrial Ca²⁺ stores nor the magnitude of [Ca²⁺]_i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP₃) generation, the Mn²⁺ influx, and the capacity of mitochondrial Ca²⁺ accumulation. Furthermore, the sensitivity of IP₃ in the Ca²⁺ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca²⁺]_i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP₃, and the slowed rate of Ca²⁺ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca²⁺ signaling cascade in NG108-15 cells.     

Controls on calcium ion fluxes in injured or shocked corn root cells: Importance of proton pumping and cell membrane potential

Passive influx of ⁴⁵Ca²⁺ into non-growing corn root tissue ( Zea mays L.) was increased as a result of actions (cutting, rubbing, chilling, heating, acidifying) or agents (cyanide, uncouplers) known to depolarize the cell membrane, and was decreased by actions (washing) or agents (fusicoccin) known to hyperpolarize it. These responses indicate the presence of Ca²⁺ channels which are voltage controlled. If the injuries were extensive, however, voltage control was lost and hyperpolarization with fusicoccin was expressed by increased ⁴⁵Ca²⁺ influx. Control could be regained by tissue washing, and millimolar levels of external Ca²⁺ would protect against loss of control. Influx of Ca²⁺ was strongly inhibited by La³⁺, but only weakly by verapamil. Intact roots showed greater cold shock sensitivity in maturing cells than in growing cells. We conclude that corn roots normally restrict Ca²⁺ influx by a mechanism linked to hyper-polarization of the plasmalemma.
Calcium ions which enter cold-shocked tissue are partially extruded during the early phase of recovery by a process stimulated by fusicoccin and subject to uncoupling.     

Calcium and plant organelles

Abstract. The role of intracellular organelles in the regulation of cytosolic Ca²⁺ levels and whether changes in these levels affect organelle metabolism is considered. We have assessed the biochemical properties of the Ca²⁺ transporting systems in mitochondrial, chloroplast and microsomal fractions. It is proposed that although all of these organelles can transport Ca²⁺ to varying extents it would appear that in some tissues at least mitochondria do not play a significant role in the maintenance of cytosolic Ca²⁺. The most important Ca²⁺ transporting systems are probably the ATP dependent Ca²⁺ extrusion across the plasma membrane and Ca²⁺ uptake by endoplasmic reticulum, as well as light driven Ca²⁺ uptake by chloroplasts. Changes in cytoplasmic [Ca²⁺] do appear to regulate the activity of NAD kinase in chloroplasts, the mitochondrial external NADH dehydrogenase and intra-mitochondrial glutamate dehydrogenase, all of which play a key role in plant cell metabolism. Since some of these enzymes are affected by primary stimuli such as light or hormones, it is concluded that Ca²⁺ may act as a second messenger mediating some of the primary responses.     

Cyclic AMP-Independent Inhibition of Voltage-Sensitive Calcium Channels by Forskolin in PC12 Cells

Abstract: Forskolin has been used to stimulate adenylyl cyclase. However, we found that forskolin inhibited voltage-sensitive Ca²⁺ channels (VSCCs) in a cyclic AMP (cAMP)-independent manner in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ was inhibited when cells were preincubated with 10 µ M forskolin. Almost maximum inhibitory effect on Ca²⁺ influx without any significant increase in cellular cAMP level was observed in PC12 cells exposed to forskolin for 1 min. In addition, the forskolin effect on Ca²⁺ influx was not affected by the presence of 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase that reduces dramatically forskolin-induced cAMP production. 1,9-Dideoxyforskolin, an inactive analogue of forskolin, also inhibited ∼80% of Ca²⁺ influx induced by 70 m M K⁺ without any increase in cAMP. The data suggest that forskolin and its analogue inhibit VSCCs in PC12 cells and that the inhibition is independent of cAMP generation.     

Salicylhydroxamic acid-stimulated NADH oxidation by purified plasmalemma vesicles from wheat roots

Purified, right side-out plasmalemma vesicles were isolated from 7-day-old roots of dark-grown wheat ( Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. The oxygen consumption by these vesicles at pH 6.5 in the presence of 1 m M NADH [12–29 nmol (mg protein)⁻¹min⁻¹] was 66% inhibited by 1 m M KCN and ca 40% by 1 m M EDTA. It was unaffected by rotenone, antimycin A, carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), mersalyl, chlorotetracycline + Ca²⁺, and EGTA. Salicylhydroxamic acid (SHAM) and its analogue, m -chlorobenzhydroxamic acid, stimulated the rate of oxygen consumption 10–20 fold in the presence of 1 m M NAD(P)H with an apparent K_m (SHAM) of ca 40 μ M (with NADH). The dependence of O₂ consumption on NADH concentration in the presence of SHAM (2 m M ) was sigmoidal, possibly due to endogenous catalase activity, and half-maximal rate was obtained at 1.5 m M . In the absence of SHAM the rate increased with increasing acidity and no pH optimum was detectable between pH 4.5 and 8.5. In the presence of SHAM an optimum was observed at pH 6.5 and 0.8 mol of H₂O₂ was produced for every 1 mol O₂ consumed. Endogenous catalase converted this H₂O₂ to O₂ and after complete conversion the stoichiometry was 2 mol NADH consumed for every mol O₃. SHAM was not consumed in the reaction. The possible involvement of a cytochrome P-450/420 system is discussed.     

Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca2+ signaling

The synaptic vesicle accumulation and subsequent morphological remodeling of axon terminals are characteristic features of presynaptic differentiation of zebrafish olfactory sensory neurons. The synaptic vesicle accumulation and axon terminal remodeling are regulated by protein kinase A and calcineurin signaling, respectively. To investigate upstream signals of presynaptic differentiation, we focused on Ca²⁺ signaling as Ca²⁺/calmodulin is required for the activation of both calcineurin and some adenylyl cyclases. We here showed that application of Ca²⁺/calmodulin inhibitor or olfactory sensory neuron-specific expression of calmodulin inhibitory peptide suppressed both synaptic vesicle accumulation and axon terminal remodeling. Thus, the trigger of presynaptic differentiation could be Ca²⁺ release from intracellular stores or Ca²⁺ influx. Application of a phospholipase C inhibitor or olfactory sensory neuron-specific expression of inositol 1,4,5-trisphosphate (IP₃) 5-phosphatase suppressed synaptic vesicle accumulation, but not morphological remodeling. In contrast, application of a voltage-gated Ca²⁺ channel blocker or expression of Kir2.1 inward rectifying potassium channel prevented the morphological remodeling. We also provided evidence that IP₃ signaling acted upstream of protein kinase A signaling. Our results suggest that IP₃-mediated Ca²⁺/calmodulin signaling stimulates synaptic vesicle accumulation and subsequent neuronal activity-dependent Ca²⁺/calmodulin signaling induces the morphological remodeling of axon terminals.     

Effect of Ca2+ on In Vitro Astrocyte Injury

Abstract: Current literature suggests that a massive influx of Ca²⁺ into the cells of the CNS induces cell damage associated with traumatic brain injury (TBI). Using an in vitro model for stretch-induced cell injury developed by our laboratory, we have investigated the role of extracellular Ca²⁺ in astrocyte injury. The degree of injury was assessed by measurement of propidium iodide uptake and release of lactate dehydrogenase. Based on results of in vivo models of TBI developed by others, our initial hypothesis was that decreasing extracellular Ca²⁺ would result in a reduction in astrocyte injury. Quite unexpectedly, our results indicate that decreasing extracellular Ca²⁺ to levels observed after in vivo TBI increased astrocyte injury. Elevating the extracellular Ca²⁺ content to twofold above physiological levels (2 m M ) produced a reduction in cell injury. The reduction in injury afforded by Ca²⁺ could not be mimicked with Ba²⁺, Mn²⁺, Zn²⁺, or Mg²⁺, suggesting that a Ca²⁺-specific mechanism is involved. Using ⁴⁵Ca²⁺, we demonstrate that injury induces a rapid influx of extracellular Ca²⁺ into the astrocyte, achieving an elevation in total cell-associated Ca²⁺ content two- to threefold above basal levels. Pharmacological elevation of intracellular Ca²⁺ levels with the Ca²⁺ ionophore A23187 or thapsigargin before injury dramatically reduced astrocyte injury. Our data suggest that, contrary to popular assumptions, an elevation of total cell-associated Ca²⁺ reduces astrocyte injury produced by a traumatic insult.     

Role of Ca2+ in Differentiation Mediated by Nerve Growth Factor and Dibutyryl Cyclic AMP in PC12 Cells

Abstract: Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca²⁺ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca²⁺ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca²⁺ influx, and Ca²⁺ mobilization from intracellular Ca²⁺ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca²⁺ accumulation by nonmitochondrial Ca²⁺ pools or the thapsigargin (TG)-sensitive Ca²⁺ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca²⁺ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca²⁺. Therefore, NGF and dbcAMP induced different effects on Ca²⁺ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca²⁺ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca²⁺ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.     

Role of external calcium in homeostasis of intracellular pH in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH

The role of external Ca²⁺ in the homeostasis of intracellular pH (pHi) of Anabaena sp. strain PCC7120 in response to a decrease in the external pH (pHex) has been studied in cell suspensions. Increase in cytoplasmic pH after acid shock is dependent on the presence of Ca²⁺ in the medium. The observed Ca²⁺-mediated alkalization of the cytoplasm depends on the extent of the shift in external pH. Acid pH shifts resulted in an increased permeability of the cytoplasmic membrane to protons, which could be reversed by increasing the concentration of Ca²⁺ in the medium. Thus, the ability of Ca²⁺ to increase cytoplasmic pH might be correlated with an inhibition of net proton uptake by increasing concentrations of external Ca²⁺ under these conditions. This combined response resulted in the generation and maintenance of a larger pH gradient (ΔpH) at acid external pH values. All Ca²⁺ channel blockers tested, such as verapamil and LaCl₃, inhibited the observed Ca²⁺-mediated response. On the other hand, the Ca ionophore calcimycin (compound A23187) was agonistic, and stimulated both cytoplasmic alkalization and inhibition of net proton uptake. The protonophorous uncoupler carbonylcyanide m -chlorophenyl hydrazone, inhibited this Ca²⁺-mediated response, whereas monensin, an inhibitor of the Na⁺/H⁺ antiporter, had no significant effect. The results of the present study suggest that an influx of Ca²⁺ from the extracellular space is required for the regulation of cytoplasmic pH in Anabaena sp. strain PCC7120 exposed to low external pH values.     

Calcium ion transport in higher plant mitochondria (Helianthus tuberosus)

A study on Ca²⁺ transport by mitochondria isolated from Jerusalem artichoke ( Helianthus tuberosus L. cv. OB1) tubers is presented. By following the distribution of Ca²⁺ under respiratory conditions, we have been able to show that Ca²⁺ accumulation into the matrix space depends on membrane potential (ΔΨ) since the uptake is not affected by the protonophore nigericin but fully blocked by valinomycin and carbonyl cyanide- p -trifluoromethoxy phenylhydrazone (FCCP). Ca²⁺ uptake requires phosphate (P_i) and is inhibited by mersalyl and by ruthenium red (RR). In addition to a Ca²⁺ influx route, mitochondria from H. tuberosus possess an RR-insensitive Ca²⁺ efflux pathway which is not stimulated by external Na⁺, Ca²⁺ is rapidly released from Ca²⁺-loaded mitochondria in the presence of ionophores such as A23187 and valinomycin and of the uncoupler FCCP. The P_i-transport inhibitor mersalyl also induces a massive Ca²⁺ release through reversal of the uptake route, the latter process being blocked by RR. Thus Jerusalem artichoke mitochondria possess a Ca²⁺ cycling mechanism which is different from that of animal mitochondria and certain other plant species.     

Mechanism Underlying the ATP-Induced Increase in the Cytosolic Ca2+ Concentration in Chick Ciliary Ganglion Neurons

Abstract: We examined the mechanism underlying the ATP-induced increase in the cytosolic Ca²⁺ concentration ([Ca]_in) in acutely isolated chick ciliary ganglion neurons, using fura-2 microfluorometry. The ATP-induced increase in [Ca]_in was dependent on external Ca²⁺, was blocked in a dose-dependent manner by reactive blue 2, and was substantially inhibited by both L- and N-type Ca²⁺ channel blockers. ATP was effective in increasing [Ca]_in in the presence of a desensitizing concentration of nicotine (100 µ M ), and simultaneous addition of maximal doses of ATP and nicotine caused an additive increase in [Ca]_in, suggesting that ATP acts on a site distinct from nicotinic acetylcholine receptors. ATP also increased the cytosolic Na⁺ concentration as determined by sodium-binding benzofuran isophthalate microfluorometry. These results suggest that ATP increases Na⁺ influx through P₂ purinoceptor-associated channels resulting in membrane depolarization, which in turn increases Ca²⁺ influx through voltage-dependent Ca²⁺ channels. However, ATP still caused a small increase in [Ca]_in under Na⁺-free conditions, and this [Ca]_in increase was little affected by Ca²⁺ channel blockers. ATP also increased Mn²⁺ influx under Na⁺-free conditions, as indicated by quenching of fura-2 fluorescence. These results suggest that nonselective cationic channels activated by ATP are permeable not only to Ca²⁺ but also to Mn²⁺, in addition to monovalent cations.     

BIOCHEMICAL CHARACTERIZATION OF THE NEURON: RIBONUCLEIC ACID COMPOSITION OF THE NEURONAL CELL BODIES

Abstract— The fluorescence of chlorotetracycline (CTC) in the presence of synaptosomes isolated from sheep brain is selectively increased by Ca²⁺ under conditions in which Mg²⁺, Na⁺, K⁺, Li⁺ or choline have only a small effect. The monovalent cations release bound Ca²⁺ from synaptosomes, and this effect is reflected by a decrease in the CTC fluorescence. Under optimal conditions there is a near parallelism between Ca²⁺ and CTC binding to the synaptosomes membranes, and Li⁺ is the monovalent cation tested which interferes the most with the binding of both substances. These results obtained in a predominantly sucrose medium become less distinct when media simulating physiological composition are utilized, which limits the usefulness of the method. Brain mitochondria and myelin also bind Ca²⁺ and CTC. The ratio of the fluorescence signal (or CTC bound) to Ca²⁺ bound is highest of all for mitochondrial membranes, and the apparent fluorescence quantum yield of CTC is also the highest in these membranes, which suggests that the Ca²⁺ in these membranes is localized in a more apolar region than is the case for synaptosomes and myelin.     

Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose

Sal1p, a novel Ca²⁺-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K _m of 0.20 ± 0.03 mM and 0.28 ± 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca²⁺ dependent. Ca²⁺ increases the V _max with a S _0.5 of 15 μM, and no changes in the K _m for ATP-Mg. Glucose sensing in yeast generates Ca²⁺ transients involving Ca²⁺ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H⁺-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca²⁺-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.     

Involvement of Protein Kinase C in Ca2+-Signaling Pathways to Activation of AP-1 DNA-Binding Activity Evoked via NMDA- and Voltage-Gated Ca2+ Channels

Abstract: Stimulation of cultured cerebellar granule cells with N -methyl- d -aspartate (NMDA) or kainic acid (KA) leads to activation of activator protein-1 (AP-1) DNA-binding activity, which can be monitored by an increase in 12- O -tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-binding activity, in concert with c- fos induction. For this increase in TRE-binding activity, Ca²⁺ influx across the plasma membrane is essential. Treatment of cells with an intracellular Ca²⁺ chelator, BAPTA-AM, abolished this increase. Close correspondence between the dose-response curves of ⁴⁵Ca²⁺ uptake and TRE-binding activity by NMDA or KA suggested that Ca²⁺ influx not only triggered sequential activation of Ca²⁺-signaling processes leading to the increase in TRE-binding activity, but also controlled its increased level. Stimulation of non-NMDA receptors by KA mainly caused Ca²⁺ influx through voltage-gated Ca²⁺ channels, whereas stimulation of NMDA receptors caused Ca²⁺ influx through NMDA-gated ion channels. The protein kinase C (PKC) inhibitors staurosporine and calphostin C inhibited the increase in TRE-binding activity caused by NMDA and KA at the same concentration at which they inhibited that caused by TPA. Furthermore, down-regulation of PKC inhibited the increase in TRE-binding activity by NMDA and KA. Thus, a common pathway that includes PKC could, at least in part, be involved in the Ca²⁺-signaling pathways for the increase in TRE-binding activity coupled with the activation of NMDA- and non-NMDA receptors.     

Mitochondria and Ca(2+) signaling

Mitochondria play a central role in cell homeostasis. Amongst others, one of the important functions of mitochondria is to integrate its metabolic response with one of the major signaling pathways - the Ca²⁺ signaling. Mitochondria are capable to sense the levels of cytosolic Ca²⁺ and generate mitochondrial Ca²⁺ responses. Specific mechanisms for both Ca²⁺ uptake and Ca²⁺ release exist in the mitochondrial membranes. In turn, the mitochondrial Ca²⁺ signals are able to produce changes in the mitochondrial function and metabolism, which provide the required level of functional integration. This essay reviews briefly the current available information regarding the mitochondrial Ca²⁺ transport systems and some of the functional consequences of mitochondrial Ca²⁺ uptake