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.     

Inhibitory effect of glutamate release from rat cerebrocortical synaptosomes by dextromethorphan and its metabolite 3-hydroxymorphinan

Dextromethorphan (DM), a widely used antitussive, has demonstrated an effective neuroprotective effect. Excessive release of glutamate is considered to be an underlying cause of neuronal damage in several neurological diseases. In the present study, we investigated whether DM or its metabolite 3-hydroxymorphinan (3-HM) could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). DM or 3-HM inhibited the Ca²⁺-dependent release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP), and this presynaptic inhibition was concentration-dependent. Inhibition of glutamate release by DM or 3-HM was resulted from a reduction of vesicular exocytosis, because the vesicular transporter inhibitor bafilomycin A1 completely blocked DM or 3-HM-mediated inhibition of 4-AP-evoked glutamate release. DM or 3-HM did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca²⁺]_C. DM or 3-HM-mediated inhibition of 4-AP-evoked glutamate release was blocked by ω-conotoxin MVIIC, an antagonist of N- and P/Q-type Ca²⁺ channel, not by dantrolene, an intracellular Ca²⁺ release inhibitor. DM or 3-HM modulation of 4-AP-evoked glutamate release appeared to involve a protein kinase C (PKC) signaling cascade, insofar as pretreatment of synaptosomes with the PKC inhibitors GF109203X or Ro318220 all effectively occluded the inhibitory effect of DM or 3-HM. Furthermore, 4-AP-induced phosphorylation of PKC was reduced by DM or 3-HM. These results suggest that DM or 3-HM inhibits glutamate release from rat cortical synaptosomes through the suppression of presynaptic voltage-dependent Ca²⁺ entry and PKC activity. This may explain the neuroprotective effects of DM against neurotoxicity.     

Fangchinoline inhibits glutamate release from rat cerebral cortex nerve terminals (synaptosomes)

Fangchinoline, an active component of radix stephaniae tetrandrinea, has been shown to possess neuroprotective properties. It has been reported that excessive glutamate release has been proposed to be involved in the pathogenesis of several neurological diseases. The primary purpose of the present study was to investigate the effect of fangchinoline on glutamate release in rat cerebral cortex nerve terminals and to explore the possible mechanism. Fangchinoline inhibited the release of glutamate evoked by 4-aminopyridine (4-AP) in a concentration-dependent manner, and this phenomenon resulted from a reduction of vesicular exocytosis but not from an inhibition of Ca²⁺-independent efflux via glutamate transporter. Fangchinoline did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca²⁺]_C. Fangchinoline-mediated inhibition of glutamate release was significantly prevented by the N- and P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC, and by the PKC inhibitors, GF109203X and Ro318220. In addition, the glutamate release mediated by direct Ca²⁺ entry with Ca²⁺ ionophore (ionomycin) was unaffected by fangchinoline, which suggests that the inhibitory effect of fangchinoline is not due to directly interfering with the release process at some point subsequent to Ca²⁺ influx. These results suggest that fangchinoline inhibits glutamate release from the rat cortical synaptosomes through the suppression of voltage-dependent Ca²⁺ channel activity and subsequent reduces Ca²⁺ entry into nerve terminals, rather than any upstream effect on nerve terminal excitability. This inhibition appears to involve the suppression of PKC signal transduction pathway. This finding may explain the neuroprotective effects of fangchinoline against neurotoxicity.     

The Effect of Spider Toxin PhTx3-4, ω-Conotoxins MVIIA and MVIIC on Glutamate Uptake and on Capsaicin-Induced Glutamate Release and [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in Spinal cord Synaptosomes

In spinal cord synaptosomes, the spider toxin PhTx3-4 inhibited capsaicin-stimulated release of glutamate in both calcium-dependent and -independent manners. In contrast, the conus toxins, ω-conotoxin MVIIA and ω-conotoxin MVIIC, only inhibited calcium-dependent glutamate release. PhTx3-4, but not ω-conotoxin MVIIA or ω-conotoxin MVIIC, is able to inhibit the uptake of glutamate by synaptosomes, and this inhibition in turn leads to a decrease in the Ca²⁺-independent release of glutamate. No other polypeptide toxin so far described has this effect. PhTx3-4 and ω-conotoxins MVIIC and MVIIA are blockers of voltage-dependent calcium channels, and they significantly inhibited the capsaicin-induced rise of intracellular calcium [Ca²⁺]_i in spinal cord synaptosomes, which likely reflects calcium entry through voltage-gated calcium channels. The inhibition of the calcium-independent glutamate release by PhTx3-4 suggests a potential use of the toxin to block abnormal glutamate release in pathological conditions such as pain.     

Partial inhibition of complex I activity increases Ca-independent glutamate release rates from depolarized synaptosomes

Mitochondria have been implicated in the pathogenesis of several neurodegenerative disorders and, in particular, complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3) activity has been shown to be partially reduced in postmortem studies of the substantia nigra of Parkinson's disease patients. The present study examines the effect of partial inhibition of complex I activity on glutamate release from rat brain synaptosomes. Following a 40% inhibition of complex I activity with rotenone, it was found that Ca²⁺-independent release of glutamate increased from synaptosomes depolarized with 4-aminopyridine. Highest rates of glutamate release were found to occur between 60–90% complex I inhibition. A similar pattern of increase was shown to occur in synaptosomes depolarized with KCl. The increase in glutamate release was found to correlate to a significant decrease in ATP. Inhibition of complex I activity by 40% was also shown to cause a significant collapse in mitochondrial membrane potential (Δ ψ _m). These results suggest that partial inhibition of complex I activity in in situ mitochondria is sufficient to significantly increase release of glutamate from the pre-synaptic nerve terminal. The relevance of these results in the context of excitotoxicity and the pathogenesis of neurodegenerative disorders is discussed.     

Functional evidence for presynaptic P2X7 receptors in adult rat cerebrocortical nerve terminals

The presynaptic P2X₇ receptor (P2X₇R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X₇R activation induced Ca²⁺-dependent vesicular glutamate release and significant Ca²⁺-independent glutamate efflux through the P2X₇R itself. In the present study, we investigated the effect of the new selective P2X₇R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca²⁺]_i signals and glutamate release, and evaluated whether P2X₇R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X₇R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X₇R-mediated [Ca²⁺]_i signals. These findings provide compelling evidence of functional presynaptic P2X₇R in cortical nerve terminals.     

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.     

Displacement of endogenous glutamate withd-aspartate: an effective strategy for reducing the calcium-independent component of glutamate release from synaptosomes

d-aspartate was used in the present study to partially deplete the cytosolic pool of glutamate, which is released independent of extracellular Ca²⁺, prior to measuring the K⁺-evoked release of this endogenous acidic amino acid from rat hippocampal mossy fiber synaptosomes. This pretreatment is known to be an effective method for substantially reducing the Ca²⁺-independent component of glutamate release. The rate of glutamate efflux is dependent on the concentration of sodium ions in the external medium and can be stimulated by exposure of hippocampal mossy fiber synaptosomes to externald-aspartate (50 M). Following the partial displacement of this cytosolic pool of glutamate withd-aspartate, the K⁺-evoked release of the residual, presumably vesicular, pool of endogenous glutamate has a strict requirement for external calcium and is highly dependent on the extent to which depolarization elevates the level of free cytosolic calcium. It is concluded that the protocol described in this study for the displacement of cytosolic glutamate withd-aspartate provides a useful alternative method of controlling for the Ca²⁺-independent component of glutamate release in synaptosomal preparations.Abbreviations used Ca calcium - Ca²⁺ free calcium - EGTA (ethylene-dioxy)diethylenedinitrilotetraacetic acid - KBM Krebs-bicarbonate medium The animals involved in this study were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council.     

Berberine Inhibits the Release of Glutamate in Nerve Terminals from Rat Cerebral Cortex

Berberine, an isoquinoline plant alkaloid, protects neurons against neurotoxicity. An excessive release of glutamate is considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. In this study, we investigated whether berberine could affect endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes) and explored the possible mechanism. Berberine inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was prevented by the chelating extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Inhibition of glutamate release by berberine was not due to it decreasing synaptosomal excitability, because berberine did not alter 4-AP-mediated depolarization. The inhibitory effect of berberine on glutamate release was associated with a reduction in the depolarization-induced increase in cytosolic free Ca²⁺ concentration. Involvement of the Ca_v2.1 (P/Q-type) channels in the berberine action was confirmed by blockade of the berberine-mediated inhibition of glutamate release by the Ca_v2.1 (P/Q-type) channel blocker ω-agatoxin IVA. In addition, the inhibitory effect of berberine on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Berberine decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synapsin I, the main presynaptic target of ERK; this decrease was also blocked by the MEK inhibition. Moreover, the inhibitory effect of berberine on evoked glutamate release was prevented in nerve terminals from mice lacking synapsin I. Together, these results indicated that berberine inhibits glutamate release from rats cortical synaptosomes, through the suppression of presynaptic Cav2.1 channels and ERK/synapsin I signaling cascade. This finding may provide further understanding of the mode of berberine action in the brain and highlights the therapeutic potential of this compound in the treatment of a wide range of neurological disorders.     

Exendin-4 Protects Mitochondria from Reactive Oxygen Species Induced Apoptosis in Pancreatic Beta Cells

Objective

Mitochondrial oxidative stress is the basis for pancreatic β-cell apoptosis and a common pathway for numerous types of damage, including glucotoxicity and lipotoxicity. We cultivated mice pancreatic β-cell tumor Min6 cell lines in vitro and observed pancreatic β-cell apoptosis and changes in mitochondrial function before and after the addition of Exendin-4. Based on these observations, we discuss the protective role of Exendin-4 against mitochondrial oxidative damage and its relationship with Ca²⁺-independent phospholipase A2.

Methods

We established a pancreatic β-cell oxidative stress damage model using Min6 cell lines cultured in vitro with tert-buty1 hydroperoxide and hydrogen peroxide. We then added Exendin-4 to observe changes in the rate of cell apoptosis (Annexin-V-FITC-PI staining flow cytometry and DNA ladder). We detected the activity of the caspase 3 and 8 apoptotic factors, measured the mitochondrial membrane potential losses and reactive oxygen species production levels, and detected the expression of cytochrome c and Smac/DLAMO in the cytosol and mitochondria, mitochondrial Ca₂-independent phospholipase A2 and Ca²⁺-independent phospholipase A2 mRNA.

Results

The time-concentration curve showed that different percentages of apoptosis occurred at different time-concentrations in tert-buty1 hydroperoxide- and hydrogen peroxide-induced Min6 cells. Incubation with 100 µmol/l of Exendin-4 for 48 hours reduced the Min6 cell apoptosis rate (p<0.05). The mitochondrial membrane potential loss and total reactive oxygen species levels decreased (p<0.05), and the release of cytochrome c and Smac/DLAMO from the mitochondria was reduced. The study also showed that Ca²⁺-independent phospholipase A2 activity was positively related to Exendin-4 activity.

Conclusion

Exendin-4 reduces Min6 cell oxidative damage and the cell apoptosis rate, which may be related to Ca²-independent phospholipase A2.     

Effect of gabaculine on metabolism and release of γ-aminobutyric acid in synaptosomes

Synaptosomes isolated from mouse brain were incubated with [¹⁴C]glutamate and [³H]-aminobutyric acid ([³H]GABA), and then [¹⁴C]GABA (newly synthesized GABA) and [³H]GABA (newly captured GABA) in the synaptosomes were analysed. (1) the [³H]GABA was rapidly degraded in the synaptosomes, (2) when the synaptosomes were treated with gabaculine (a potent inhibitor of GABA aminotransferase), the degradation of [³H]GABA was strongly inhibited, (3) the gabaculine treatment brough about a significant increase in Ca²⁺-independent release of [³H]GABA with no effect on Ca²⁺-dependent release, (4) no effects of gabaculine on degradation and release of [¹⁴C]GABA were observed. The results indicate that there are at least two pools of GABA in synaptosomes and support the possibilities that GABA taken up into a pool which is under the influence of GABA aminotransferase is released Ca²⁺-independently and that GABA synthesized in another pool which is not under the influence of GABA aminotransferase is released Ca²⁺-dependently.     

Effects of dexamethasone on K(+)-evoked glutamate release from rat hippocampal slices

Dexamethasone (DEX) at physiologically elevated (stress) concentration (1 µM) decreased K⁺-evoked glutamate release from rat hippocampal slices under superfusion in the presence of Ca²⁺. On the contrary 10 µM DEX increased this K⁺-evoked glutamate release while 0.1 µM DEX had no effect. The glucocorticoid antagonist for the classic receptor, RU 486, completely reversed the effect of 1 µM DEX. Actinomycin D had no effect. Dexamethasone at 1 µM had no effect on the Ca²⁺-independent (10 µM Mg²⁺ replacing 1 mM Ca²⁺) K⁺-evoked glutamate release. Dexamethasone at 1 µM or 10 µM had no effect on the phosphate-activated glutaminase—the key enzyme for the biosynthesis of neurotransmitter glutamate. These results suggest that the effect of DEX on K⁺-evoked glutamate release: (i) depends on its concentration; (ii) is exerted on the Ca²⁺-dependent (neurotransmitter release), at least at physiological stress concentrations; and (iii) is exerted via the classical receptor but is nongenomic.     

The release of glutamate and aspartate from rat brain synaptosomes in response to domoic acid (amnesic shellfish toxin) and kainic acid

Kainic acid is known to stimulate the release of glutamate (GLU) and aspartate (ASP) from presynaptic neurons. It has been suggested that the enhanced release of these endogenous EAA's plays a significant role in the excitotoxic effects of KA. Domoic acid (DOM), a shellfish toxin, is structurally similar to KA, and has been shown to be 3–8 times more toxic than KA. In this study, effects of KA and DOM on the release of GLU and ASP from rat brain synaptosomes were investigated. Amino acid analysis was performed by the reversed phase HPLC, following derivatization with 9-fluorenylmethyl chloroformate (FMOC). Potassium chloride (40 mM) was used as a positive control, and stimulated GLU release from rat brain synaptosomes in presence or absence of Ca²⁺. DOM enhanced the release of GLU, whereas KA stimulated the release of both GLU and ASP from synaptosomes in the presence of Ca²⁺. However, their potency to stimulate GLU and ASP release was enhanced in absence of Ca²⁺. These results indicate that diferent mechanisms may be involved in the release of GLU and ASP in response to DOM and KA, and that neurotransmitter release appeared to be highly specific for these agonists. It would appear that DOM and KA may interact with different receptors on the presynaptic nerve terminal, and/or activate different subtypes of voltage-dependent Ca²⁺ channels to promote influx of Ca²⁺ which is targeted for different pools of neurotransmitters.Abbreviations ANOVA analysis of variance - ASP aspartate - DOM domoic acid - DHKA dihydrokainic acid - EAA excitatory amino acid - FMOC 9-fluorenylmethyl chloroformate - GLU glutamate - KA kainic acid     

Pre‐synaptic kainate receptor‐mediated facilitation of glutamate release involves PKA and Ca2+‐calmodulin at thalamocortical synapses

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 μM) produced an increase in 4‐aminopyridine (4‐AP)‐evoked glutamate release. In thalamocortical slices, KA (1 μM) produced an increase in the amplitude of evoked excitatory post‐synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, and persisted after pre‐treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G‐protein‐independent regulation further in thalamocortical slices, the KAR‐mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca²⁺ permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca²⁺ stores by thapsigargin, or inhibition of Ca²⁺‐induced Ca²⁺‐release by ryanodine. Thus, the KA‐mediated modulation was contingent on both Ca²⁺ entry through Ca²⁺‐permeable KARs and liberation of intracellular Ca²⁺ stores. Finally, sensitivity to W‐7 indicated that the increased cytosolic [Ca²⁺] underpinning KAR‐mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre‐synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca²⁺‐calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G‐protein involvement.

     

Atypical properties of a conventional calcium channel β subunit from the platyhelminth Schistosoma mansoni

Background

Serotonin induces fluid secretion from Calliphora salivary glands by the parallel activation of the InsP₃/Ca²⁺ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca²⁺ signaling and InsP₃-induced Ca²⁺ release from the endoplasmic reticulum (ER).

Results

Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca²⁺]_i changes into a sustained increase. Intraluminal Ca²⁺ measurements in the ER of β-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP₃-induced Ca²⁺ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP₃ receptor Ca²⁺ channel for InsP₃. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP₃-induced Ca²⁺ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP₃/Ca²⁺ signaling pathway for 5-HT, because IBMX potentiated Ca²⁺-dependent Cl^- transport activated by a threshold 5-HT concentration.

Conclusion

This report shows, for the first time for an insect system, that cAMP can potentiate InsP₃-induced Ca²⁺ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP₃/Ca²⁺ signaling pathways enhances transepithelial electrolyte transport.     

Acute Restraint Stress Enhances Calcium Mobilization and Glutamate Exocytosis in Cerebrocortical Synaptosomes from Mice

Acute stress is known to enhance the memory of events that are potentially threatening to the organisms. Glutamate, the most abundant excitatory neurotransmitter in the mammalian central nervous system, plays a critical role in learning and memory formation and calcium (Ca²⁺) plays an essential role in transmitter release from nerve terminals (synaptosomes). In the present study, we investigated the effects of acute restraint stress on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in cerebrocortical synaptosomes from mice. Acute restraint stress caused a significant increase in resting [Ca²⁺]_i and significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. It also brought about a significant increase in spontaneous (unstimulated) glutamate release and significantly enhanced K⁺- and 4-AP-induced Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) completely suppressed the enhancements of [Ca²⁺]_i and Ca²⁺-dependent glutamate release in acute restraint-stressed mice. These results indicate that acute restraint stress enhances K⁺- or 4-AP-induced glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through P- and N-type Ca²⁺ channels.     

Presynaptic External Calcium Signaling Involves the Calcium-Sensing Receptor in Neocortical Nerve Terminals

Background

Nerve terminal invasion by an axonal spike activates voltage-gated channels, triggering calcium entry, vesicle fusion, and release of neurotransmitter. Ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry. Consequently characterization of the functional properties of ion channels at nerve terminals is crucial to understand the regulation of transmitter release. Direct recordings from small neocortical nerve terminals have revealed that external [Ca²⁺] ([Ca²⁺]_o) indirectly regulates a non-selective cation channel (NSCC) in neocortical nerve terminals via an unknown [Ca²⁺]_o sensor. Here, we identify the first component in a presynaptic calcium signaling pathway.

Methodology/Principal Findings

By combining genetic and pharmacological approaches with direct patch-clamp recordings from small acutely isolated neocortical nerve terminals we identify the extracellular calcium sensor. Our results show that the calcium-sensing receptor (CaSR), a previously identified G-protein coupled receptor that is the mainstay in serum calcium homeostasis, is the extracellular calcium sensor in these acutely dissociated nerve terminals. The NSCC currents from reduced function mutant CaSR mice were less sensitive to changes in [Ca²⁺]_o than wild-type. Calindol, an allosteric CaSR agonist, reduced NSCC currents in direct terminal recordings in a dose-dependent and reversible manner. In contrast, glutamate and GABA did not affect the NSCC currents.

Conclusions/Significance

Our experiments identify CaSR as the first component in the [Ca²⁺]_o sensor-NSCC signaling pathway in neocortical terminals. Decreases in [Ca²⁺]_o will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca²⁺]_o following its entry via voltage-activated Ca²⁺ channels. CaSR may detects such falls in [Ca²⁺]_o and increase action potential duration by increasing NSCC activity, thereby attenuating the impact of decreases in [Ca²⁺]_o on release probability. CaSR is positioned to detect the dynamic changes of [Ca²⁺]_o and provide presynaptic feedback that will alter brain excitability.     

Exposure to Ebselen Changes Glutamate Uptake and Release by Rat Brain Synaptosomes

We investigated effects of Ebselen, diphenyl diselenide (PhSe)₂ and diphenyl ditelluride (PhTe)₂ on [³H]glutamate uptake and release by brain synaptosomes. Ebselen after acute exposure inhibited K⁺-stimulated [³H]glutamate release by brain synaptosomes. (PhSe)₂ and (PhTe)₂ did not change [³H]glutamate release by brain synaptosomes. Ebselen, (PhSe)₂ and (PhTe)₂ had no significantly effects on [³H]glutamate uptake after acute exposure. In vitro, Ebselen (100 M) inhibited [³H]glutamate release and uptake. (PhSe)₂ had no significant effect, while (PhTe)₂ (100 M) inhibited [³H]glutamate uptake by brain synaptosomes. In vitro, (PhSe)₂, (PhTe)₂ and Ebselen caused a significant inhibition of [³H]glutamate uptake by brain synaptic vesicles in vitro. The results demonstrated that organochalcogenides have a rather complex effect on glutamate homeostasis depending on the compound and the schedule of exposition. We propose that the neuroprotective action of Ebselen can be related, in addition to its glutathione peroxidase-like and antilipoperoxidative activity, to a direct interaction with the glutamatergic system by reducing K^ï-evoked glutamate release.     

Protocol for the BAG-RECALL clinical trial: a prospective, multi-center, randomized, controlled trial to determine whether a bispectral index-guided protocol is superior to an anesthesia gas-guided protocol in reducing intraoperative awareness with explicit recall in high risk surgical patients

Background

Sevoflurane has been demonstrated to vasodilate the foeto-placental vasculature. We aimed to determine the contribution of modulation of potassium and calcium channel function to the vasodilatory effect of sevoflurane in isolated human chorionic plate arterial rings.

Methods

Quadruplicate ex vivo human chorionic plate arterial rings were used in all studies. Series 1 and 2 examined the role of the K⁺ channel in sevoflurane-mediated vasodilation. Separate experiments examined whether tetraethylammonium, which blocks large conductance calcium activated K⁺ (K_Ca++) channels (Series 1A+B) or glibenclamide, which blocks the ATP sensitive K⁺ (K_ATP) channel (Series 2), modulated sevoflurane-mediated vasodilation. Series 3 – 5 examined the role of the Ca⁺⁺ channel in sevoflurane induced vasodilation. Separate experiments examined whether verapamil, which blocks the sarcolemmal voltage-operated Ca⁺⁺ channel (Series 3), SK&F 96365 an inhibitor of sarcolemmal voltage-independent Ca⁺⁺ channels (Series 4A+B), or ryanodine an inhibitor of the sarcoplasmic reticulum Ca⁺⁺ channel (Series 5A+B), modulated sevoflurane-mediated vasodilation.

Results

Sevoflurane produced dose dependent vasodilatation of chorionic plate arterial rings in all studies. Prior blockade of the K_Ca++ and K_ATP channels augmented the vasodilator effects of sevoflurane. Furthermore, exposure of rings to sevoflurane in advance of TEA occluded the effects of TEA. Taken together, these findings suggest that sevoflurane blocks K⁺ channels. Blockade of the voltage-operated Ca⁺⁺channels inhibited the vasodilator effects of sevoflurane. In contrast, blockade of the voltage-independent and sarcoplasmic reticulum Ca⁺⁺channels did not alter sevoflurane vasodilation.

Conclusion

Sevoflurane appears to block chorionic arterial K_Ca++ and K_ATP channels. Sevoflurane also blocks voltage-operated calcium channels, and exerts a net vasodilatory effect in the in vitro foeto-placental circulation.     

Programmed cell death of secretory cavity cells of citrus fruits is associated with Ca2+ accumulation in the nucleus

Key message

An increase in Ca ²⁺ concentration in the nucleus may activate the PCD of secretory cavity cells, and further Ca ²⁺ accumulation contributes to the regulation of nuclear DNA degradation.

Abstract

Calcium plays an important role in plant programmed cell death (PCD). Previously, we confirmed that PCD was involved in the degradation of secretory cavity cells in Citrus sinensis (L.) Osbeck fruits. To further explore the function of calcium in the PCD of secretory cavity cells, we used potassium pyroantimonate precipitation to detect and locate calcium dynamics. At the precursor cell stage of the secretory cavity, Ca²⁺ was only distributed in the cell walls. At the early stage of secretory cavity initial cells, Ca²⁺ in the cell walls was gradually transported into the cytoplasm via pinocytotic vesicles. Although a small amount of Ca²⁺ was present in the nucleus, the TUNEL signal was scarcely observed. At the middle stage of initial cells, a large number of pinocytotic vesicles were transferred to the nucleus, where the vesicle membrane fused with the nuclear membrane to release calcium into the nucleoplasm. In addition, abundant Ca²⁺ aggregated in the condensed chromatin and nucleolus, where the TUNEL signal appeared the strongest. At the late stage of initial cells, the chromatin and nucleolus gradually degraded and disappeared, and the nucleus appeared broken-like, as Ca²⁺ in the cell wall had nearly completely disappeared, and Ca²⁺ in the nucleus was also rapidly reduced. Furthermore, the TUNEL signal also disappeared. These phenomena indicated that an increase in Ca²⁺ concentration in the nucleus might activate the PCD of secretory cavity cells, and further Ca²⁺ accumulation contributed to the regulation of nuclear DNA degradation.