Calcium-Dependent and-Independent Release of Glutamate from Synaptosomes Monitored by Continuous Fluorometry

An enzyme-linked fluorometric assay is described for the continuous monitoring of the unidirectional efflux of glutamate from guinea-pig synaptosomes. Glutamate efflux from freshly suspended, polarized synaptosomes occurs at 0.35-0.39 nmol min-1 mg of protein-1 and is not significantly affected by external Ca2+. KCl depolarization (30 mMKCl) in the absence of Ca2+ doubles this rate, whereas in the presence of Ca2+, the initial kinetics of the assay are consistent with the release in the first 5 s of 0.6 nmol mg of protein-1. The final extent of Ca2+-dependent release amounts to 1.9 nmol mg of protein-1, or 8.5% of the total intrasynaptosomal glutamate content. Preincubation of synaptosomes at 30 degrees C for 2 h before depolarization leads to a decrease in Ca2+-independent release and an increase in Ca2+-dependent release, consistent with an intrasynaptosomal relocation of the amino acid.     

Glutamate release by an Na+ load and oxidative stress in nerve terminals: relevance to ischemia/reperfusion

Previously we have reported that oxidative stress induced by hydrogen peroxide exacerbates the effect of an Na+ load in isolated nerve terminals, with a consequence of an ATP depletion, [Ca2+]i and [Na+]i deregulation, and collapse of mitochondrial membrane potential. In the present study, the release of glutamate in response to a combined effect of an [Na+] load and oxidative stress was measured in isolated nerve terminals over an incubation for 15 min. Exposure to hydrogen peroxide (100 micro m) had no effect on the release of glutamate, but significantly enhanced the Ca2+-independent glutamate release induced by a small [Na+] load achieved with 10 micro m veratridine. The effect of a larger Na+ load induced by 40 micro m veratridine was not further increased by hydrogen peroxide; in contrast the external Ca2+-dependent glutamate release was completely eliminated by the oxidant under this condition. The effects of oxidative stress superimposed on a Na+ load are consistent with at least two factors: (i) a relatively modest Na+ load induced by veratridine is augmented by H2O2 giving rise to an increased Ca2+-independent release of glutamate (ii) oxidative stress in combination with a larger Na+ load causes severe ATP depletion limiting the Ca2+-dependent vesicular glutamate release. Given the concurrent presence of an Na+ load and oxidative stress in ischemia/reperfusion these results indicate that the extent of the Na+ load developing during the ischemic period could determine the release of glutamate induced by an oxidative stress during reperfusion.     

Glutamine and Aspartate Loading of Synaptosomes: A Reevaluation of Effects on Calcium-Dependent Excitatory Amino Acid Release

Guinea-pig cerebral cortical synaptosomes were preincubated for 60 min with 100 microM D-aspartate, L-aspartate, or L-glutamate. The total D- plus L-aspartate content of the synaptosomal fraction increased to 235%, 195%, or 164%, respectively, of the control. Despite this no increase was seen in the very low KCl evoked, Ca2+-dependent release of aspartate. Preincubation with the three amino acids changed the synaptosomal glutamate content to 78% (D-aspartate), 149% (L-aspartate), or 168% (L-glutamate) of control. However there was no statistically significant effect of these preincubations on the extent of Ca2+-dependent glutamate release. Thus the Ca2+-dependent release of aspartate and glutamate is not determined by the total synaptosomal content of these amino acids. The addition of 0.1-0.5 mM glutamine to the incubation caused a massive appearance of glutamate in the extrasynaptosomal medium. Analysis of specific activities showed that glutamine was hydrolysed directly by an extrasynaptosomal glutaminase, and that intrasynaptosomal glutamate was predominantly labelled by uptake of this glutaminase-derived glutamate. No increase was seen in the extent of Ca2+-dependent release of glutamate (by fluorimetry) either after preincubation with glutamine or in the continued presence of glutamine. Thus we are unable to confirm reports that glutamine expands the transmitter pool of glutamate. The extrasynaptosomal glutaminase activity in the synaptosomal preparation was inhibited by Ca2+ and activated by phosphate. Identical kinetics were obtained with "free" brain mitochondria, confirming the origin of the glutamine-derived glutamate.     

PnTx3-6 a spider neurotoxin inhibits K+-evoked increase in [Ca2+](i) and Ca2+-dependent glutamate release in synaptosomes

The present experiments investigated the effect of a neurotoxin purified from the venom of the spider Phoneutria nigriventer. This toxic component, P. nigriventer toxin 3-6 (PnTx3-6), abolished Ca(2+)-dependent glutamate release with an IC(50) of 74.4nM but did not alter Ca(2+)-independent secretion of glutamate when brain cortical synaptosomes were depolarized by KCl (33mM). This effect was most likely due to interference with the entry of calcium through voltage activated calcium channels (VACC), reducing the increase in the intrasynaptosomal free calcium induced by membrane depolarization with an IC(50) of 9.5nM. We compared the alterations induced by PnTx3-6 with the actions of toxins known to block calcium channels coupled to exocytosis. Our results indicate that PnTx3-6 inhibition of glutamate release and intrasynaptosomal calcium involves P/Q type calcium channels and this toxin can be a valuable tool in the investigation of calcium channels.     

Ca2+-Independent Release of Glutamate During In Vitro Anoxia in Isolated Nerve Terminals

The effects of in vitro anoxia on the release of glutamate in isolated nerve terminals were studied. The extra-synaptosomal concentration of glutamate ([Glu]ext) under aerobic conditions was 2.3 microM and increased to 4.9 microM after 10 min of anoxia. However, when synaptosomes were incubated in the presence of lactate plus pyruvate instead of glucose, to prevent anaerobic glycolysis, anoxia induced an eightfold increase in the [Glu]ext. The accumulation of glutamate in the external medium during anoxia was Ca2+ independent and insensitive to a significant reduction of the Ca(2+)-dependent release of the amino acid. These results indicate that a Ca(2+)-independent efflux of cytoplasmic glutamate occurs during in vitro anoxia in isolated nerve terminals.     

4-Aminobutyrate Can Be Released Exocytotically from Guinea-Pig Cerebral Cortical Synaptosomes

Guinea-pig synaptosomes possess two functional pools of 4-aminobutyrate (GABA). One is rapidly labelled by added [14C]GABA, is steadily released in a Ca2+-independent manner when the Na+ electrochemical potential across the plasma membrane is collapsed, and is depleted by the GABA analogue 2,4-diaminobutyrate (DABA), all of which is consistent with a cytosolic location. A second, noncytosolic compartment only slowly equilibrates with exogenous [14C]GABA, is not depleted by DABA, but can release 350 pmol of endogenous GABA/mg of protein (8% of the total intrasynaptosomal GABA) within 15 s of depolarization in the presence of Ca2+. Ca2+-independent release occurs by thermodynamic reversal of the plasma membrane uptake pathway following artifactually prolonged depolarization, whereas Ca2+-dependent release is consistent with physiological exocytosis from vesicular stores.     

Glutamate Release from Guinea-Pig Synaptosomes: Stimulation by Reuptake-Induced Depolarization

Glutamate (10-100 microM) reversibly depolarizes guinea-pig cerebral cortical synaptosomes. This does not appear to be because of a conventional autoreceptor. Neither kainate at 1 mM, 100 microM N-methyl-D-aspartate (NMDA), 100 microM L-2-amino-4-phosphonobutanoate (APB), nor 100 microM quisqualate affects the Ca2+-dependent release of glutamate from suboptimally depolarized synaptosomes. However, kainate, quisqualate, and the quisqualate agonists beta-N-oxalylamino-L-alanine and alpha-amino-3-hydroxy-5-methylisoxazole propionate cause a slow Ca2+-independent release of glutamate from polarized synaptosomes. However, unlike kainate, quisqualate does not inhibit the acidic amino acid carrier. APB, NMDA, and the NMDA receptor-mediated neurotoxin beta-N-methylamino-L-alanine do not influence Ca2+-independent release at 100 microM. The depolarization of the plasma membrane by glutamate can be mimicked by D-aspartate, can be blocked by the transport inhibitor dihydrokainate, and is accompanied by the net uptake of acidic amino acids. L-Glutamate or D-aspartate at 100 microM increases the cytoplasmic free Ca2+ concentration. D-aspartate at 100 microM causes a Ca2+-dependent release of endogenous glutamate, superimposed on the Ca2+-independent heteroexchange with glutamate through the acidic amino acid carrier. The results suggest that the glutamatergic subpopulation of synaptosomes can be depolarized by exogenous glutamate.     

Ca2+-Dependent Regulation of Presynaptic Stimulus-Secretion Coupling

In the present study, we have investigated the role of Ca2+ in the coupling of membrane depolarization to neurotransmitter secretion. We have measured (a) intracellular free Ca2+ concentration ([Ca2+]i) changes, (b) rapid 45Ca2+ uptake, and (c) Ca2+-dependent and -independent release of endogenous glutamate (Glu) and gamma-aminobutyric acid (GABA) as a function of stimulus intensity by elevating the extracellular [K+] to different levels in purified nerve terminals (synaptosomes) from rat hippocampus. During stimulation, Percoll-purified synaptosomes show an increased 45Ca2+ uptake, an elevated [Ca2+]i, and a Ca2+-dependent as well as a Ca2+-independent release of both Glu and GABA. With respect to both amino acids, synaptosomes respond on stimulation essentially in the same way, with maximally a fourfold increase in Ca2+-dependent (exocytotic) release. Ca2+-dependent transmitter release as well as [Ca2+]i elevations show maximal stimulation at moderate depolarizations (30 mM K+). A correlation exists between Ca2+-dependent release of both Glu and GABA and elevation of [Ca2+]i. Ca2+-dependent release is maximally stimulated with an elevation of [Ca2+]i of 60% above steady-state levels, corresponding with an intracellular concentration of approximately 400 nM, whereas elevations to 350 nM are ineffective in stimulating Ca2+-dependent release of both Glu and GABA. In contrast, Ca2+-independent release of both Glu and GABA shows roughly a linear rise with stimulus intensity up to 50 mM K+. 45Ca2+ uptake on stimulation also shows a continuous increase with stimulus intensity, although the relationship appears to be biphasic, with a plateau between 20 and 40 mM K+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repetitive Action Potentials in Isolated Nerve Terminals in the Presence of 4-Aminopyridine: Effects on Cytosolic Free Ca2+ and Glutamate Release

The mechanisms by which an elevated KCl level and the K+-channel inhibitor 4-aminopyridine induce release of transmitter glutamate from guinea-pig cerebral cortical synaptosomes are contrasted. KCl at 30 mM caused an initial spike in the cytosolic free Ca2+ concentration ([Ca2+]c), followed by a partial recovery to a plateau 112 +/- 13 nM above the polarized control. The Ca2+-dependent release of endogenous glutamate, determined by continuous fluorimetry, was largely complete by 3 min, by which time 1.70 +/- 0.35 nmol/mg was released. [Ca2+]c elevation and glutamate release were both insensitive to tetrodotoxin. KCl-induced elevation in [Ca2+]c could be observed in both low-Na+ medium and in the presence of low concentrations of veratridine. 4-Aminopyridine at 1 mM increased [Ca2+]c by 143 +/- 18 nM to a plateau similar to that following 30 mM KCl. The initial rate of increase in [Ca2+]c following 4-aminopyridine administration was slower than that following 30 mM KCl, and a transient spike was less apparent. Consistent with this, the 4-aminopyridine-induced net uptake of 45Ca2+ is much lower than that following an elevated KCl level. 4-Aminopyridine induced the Ca2+-dependent release of glutamate, although with somewhat slower kinetics than that for KCl. The measured release was 0.81 nmol of glutamate/mg in the first 3 min of 4-aminopyridine action. In contrast to KCl, glutamate release and the increase in [Ca2+]c with 4-aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na+ channels. Basal [Ca2+]c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Facilitatory effect of glutamate exocytosis from rat cerebrocortical nerve terminals by alpha-tocopherol, a major vitamin E component

The effect of alpha-tocopherol, the major vitamin E component, on the release of endogenous glutamate has been investigated using rat cerebrocortical nerve terminals. Results showed that alpha-tocopherol facilitated the Ca2+-dependent but not the Ca2+-independent glutamate release evoked by 4-aminopyridine (4AP). This release facilitation was insensitive to glutamate transporter inhibitor L-trans-PDC or DL-TBOA, and blocked by the exocytotic neurotransmitter release inhibitor tetanus neurotoxin, indicating that alpha-tocopherol affects specifically the physiological exocytotic vesicular release without affecting the non-vesicular release. Facilitation of glutamate exocytosis by alpha-tocopherol was not due to its increasing synaptosomal excitability, because alpha-tocopherol did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential. Rather, examination of the effect of alpha-tocopherol on cytoplasmic free Ca2+ concentration revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca2+ influx. Consistent with this, the alpha-tocopherol-mediated facilitation of glutamate release was significantly reduced in synaptosomes pretreated with omega-CgTX MVIIC, a wide spectrum blocker of N- and P/Q-type Ca2+ channels. In addition, alpha-tocopherol modulation of glutamate release appeared to involve a protein kinase C (PKC) signalling cascade, insofar as pretreatment of synaptosomes with the PKC inhibitor GF109203X effectively suppressed the facilitatory effect of alpha-tocopherol on 4AP- or ionomycin-evoked glutamate release. Furthermore, alpha-tocopherol increased the phosphorylation of MARCKS, the major presynapic substrate for PKC, and this effect was also significantly attenuated by PKC inhibition. Together, these results suggest that alpha-tocopherol exerts an increase in PKC activation, which subsequently enhances voltage-dependent Ca2+ influx and vesicular release machinery to cause an increase in evoked glutamate release from rat cerebrocortical glutamatergic terminals. This finding might provide important information regarding to the action of vitamin E in the central nervous system.     

Kainic Acid Inhibits the Synaptosomal Plasma Membrane Glutamate Carrier and Allows Glutamate Leakage from the Cytoplasm but Does Not Affect Glutamate Exocytosis

Kainate inhibits the exchange of D-aspartate into guinea-pig cerebrocortical synaptosomes. Kainate inhibits the Ca2+-independent efflux of endogenous glutamate in the presence of a trapping system for the released amino acid but potentiates a Ca2+-independent net efflux of endogenous and labelled glutamate and aspartate in the absence of the trap. Dihydrokainate has a similar effect. No discrepancy is seen between the release of endogenous and exogenously accumulated amino acid. These results are consistent with the presence of a slow leak of glutamate or aspartate from the cytoplasm independent of the kainate-sensitive Na+-cotransport pathway. In the presence of the trap, glutamate effluxes by both pathways, whereas in the absence of the trap, the Na+-cotransport pathway opposes the leak. Neither in the presence or absence of the glutamate trap does kainate induce, inhibit, or otherwise affect the Ca2+-dependent release of endogenous glutamate. The results enable many of the apparent complexities in the presynaptic actions of kainate to be resolved.     

Mechanisms of glutamate release elicited in rat cerebrocortical nerve endings by 'pathologically' elevated extraterminal K+ concentrations

Extracellular [K+] can increase during some pathological conditions, resulting into excessive glutamate release through multiple mechanisms. We here investigate the overflow of [3H]D-aspartate ([3H] D-ASP) and of endogenous glutamate elicited by increasing [K+] from purified rat cerebrocortical synaptosomes. Depolarization with [K+] 15 mmol/L were prevented by the glutamate transporter inhibitors DL-threo-beta-benzyloxyaspartate (DL-TBOA) and dihydrokainate. Differently, the overflows of endogenous glutamate provoked by [K+] > 15 mmol/L were insensitive to both inhibitors; the external Ca2+-independent glutamate overflow caused by 50 mmol/L KCl was prevented by bafilomycin, by chelating intraterminal Ca2+, by blocking the mitochondrial Na+/Ca2+ exchanger and, for a small portion, by blocking anion channels. In contrast to purified synaptosomes, the 50 mmol/L K+-evoked release of endogenous glutamate or [3H]D-ASP was inhibited by DL-TBOA in crude synaptosomes; moreover, it was external Ca2+-insensitive and blocked by DL-TBOA in purified gliosomes, suggesting that carrier-mediated release of endogenous glutamate provoked by excessive [K+] in CNS tissues largely originates from glia.     

Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes

The exocytotic release of L-glutamate from guinea-pig cerebral cortical synaptosomes can be extensively inhibited by preincubation with botulinum neurotoxin type A at 37 degrees C for 1-2 h. The toxin has no effect on synaptosomal respiratory control, respiratory capacity, ATP synthesis, plasma-membrane 86Rb+ permeability or plasma-membrane potential, does not inhibit the entry of 45Ca2+ into the synaptosome upon depolarization and does not alter the ability of intrasynaptosomal mitochondria to sequester Ca2+. The blockade of Ca2+-dependent glutamate release may be totally reversed by the Ca2+/2 H+-exchange ionophore ionomycin, but not by increasing extracellular Ca2+ concentration. It is suggested (a) that exocytosis is triggered by the penetration of Ca2+ into an intracellular hydrophobic milieu; (b) that this stage is blocked by the toxin and (c) that ionomycin is able to bypass this block and deliver Ca2+ to the exocytotic apparatus.     

Relation of Acetylcholine Release to Ca2+ Uptake and Intraterminal Ca2+ Concentration in Guinea-Pig Cortex Synaptosomes

[14C]Acetylcholine (ACh) release and parallel alterations in 45Ca2+ uptake and intrasynaptosomal free CA2+ concentration ([Ca2+]i) were measured in guinea-pig brain cortex synaptosomes. Depolarization by high K+ concentrations caused a rapid transient increase in Ca2+ uptake, terminating within 60 s (rate constant = 0.060 s-1; t1/2 = 11.6 s). This resulted in a rapid increase (within 1 s) in [Ca2+1]i, which then fell to a maintained but still-elevated plateau level (t1/2 for the decline was 15 s). Peaks of [Ca2+]i showed a sigmoidal dependence on depolarization, contrasting with the simple linear dependence of plateau levels of [Ca2+]i. The K+-evoked ACh release also had two phases: a fast initial increase (t1/2 = 11.3 s), which terminated within 60 s, was followed by a slow additional increase during sustained depolarizations of up to 10 min. Depolarization by veratridine led to a slow gradual increase in Ca2+ uptake (t1/2 = 130 s) over a 10-min incubation period, whereas an elevated plateau level of [Ca2+]i was achieved within 2 min (without a rapid peak elevation). The Ca2+-dependent fraction of the veratridine-evoked ACh release correlated with the increase in [Ca2+]i rather than with Ca2+ uptake. Using two different methods of depolarization partially circumvented the time limitations imposed by a buffering Ca2+ indicator and we suggest that, in the main, ACh is released in bursts associated with [Ca2+]i transients.     

Effect of Portacaval Anastomosis on Electrically Stimulated Release of Glutamate from Rat Hippocampal Slices

To evaluate the effects of chronic liver failure on release of the excitatory transmitter glutamate, electrically stimulated Ca2(+)-dependent and Ca2(+)-independent release of glutamate in the absence or presence of NH4+ was studied in superfused slices of hippocampus from portacaval-shunted or sham-operated rats 4 weeks after surgery. Spontaneous and stimulation-evoked release of glutamate was higher in shunted rats in the presence of normal or low Ca2+ concentrations, and this release was depressed by 5 mM ammonium chloride. These findings suggest that portacaval shunting results in increased levels of extracellular glutamate in brain, probably due to a decreased reuptake of glutamate into perineuronal astrocytes, shown in previous studies to undergo neuropathological changes following portacaval shunting. Changes in the inactivation of transmitter glutamate could be responsible, at least in part, for the neurological dysfunction resulting from sustained hyperammonemia and portal-systemic shunting resulting from chronic liver failure.     

Mutual Antagonism of κ-Opiate and α2-Adrenoceptor Agonist Effects on Intrasynaptosomal Free [Ca2+]i

Synaptosomes prepared from rat cerebral cortices on Percoll discontinuous density gradients were loaded with the fluorescent EGTA analogue Quin 2 to allow measurement of intracellular free [Ca2+]i. When either kappa-opiate or alpha 2-adrenoceptor agonists were incubated with the synaptosomes, there was a highly significant (p less than 0.004, p less than 2.7 X 10(-6), respectively) reduction in intrasynaptosomal free [Ca2+]i relative to controls. As these synaptosomes are not depolarised, the data suggest that both alpha 2-adrenoceptor agonists and kappa-opiate agonists inhibit neurotransmitter release, decreasing the availability of intraneuronal [Ca2+]i rather than altering Ca2+ entry. However, when these two agonists were coincubated, there was a complete abolition of the effects of either agonist; in fact, there was an apparent increase in the intrasynaptosomal free [Ca2+]i. Neither morphine nor [D-Ala2-D-Leu5]enkephalin, mu and delta opiate agonists respectively, had any significant effect on intrasynaptosomal free [Ca2+]i. These results show that the individual effects of clonidine and dynorphin A1-13 are in keeping with the role of these substances at autoreceptors controlling neurotransmitter release. The mutual antagonism of their effects on [Ca2+]i is more difficult to explain but it may be a mechanism that prevents the occurrence of excessive inhibition of neuronal systems.     

Inhibition of Glutamate Release by Arachidonic Acid in Rat Cerebrocortical Synaptosomes

The action of arachidonic acid on glutamate release in rat cerebrocortical synaptosomes was investigated. The Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4-AP) was inhibited by arachidonic acid (0.5-10 microM), but the KCl-evoked release was not modified. The Ca(2+)-independent release of glutamate was insensitive to low concentrations of arachidonic acid, but higher concentrations of this free fatty acid (30 microM) induced a slow efflux of cytoplasmic glutamate. The decrease in the Ca(2+)-dependent release of glutamate by arachidonic acid was consistent with a reduction in both the depolarization and the subsequent rise in the cytoplasmic free Ca2+ concentration induced by 4-AP in the nerve terminal. The inhibitory action by arachidonic acid observed in glutamate release was reversed in the presence of the K(+)-channel blocker tetraethylammonium.     

Calmidazolium selectively inhibits exocytotic glutamate release evoked by P2X7 receptor activation

We previously observed that activation of presynaptic P2X7 receptors located on rat cerebrocortical nerve terminals induced the release of glutamate through different modes: the channel conformation allowing Ca(2+) entry triggered exocytotic release, while the receptor itself functioned as a permeation pathway for the non-exocytotic glutamate release. Considering that exocytotic and non-exocytotic glutamate release evoked by the activation of P2X7 receptors might play a role in the control of glutamatergic synapses, we investigated whether calmidazolium (which has been found to inhibit small cation currents through recombinant P2X7 receptors, but not organic molecule permeation) could distinguish between P2X7-related exocytotic and non-exocytotic modes of glutamate release. We found that calmidazolium inhibited the intrasynaptosomal Ca(2+) response to P2X7 receptor activation and the Ca(2+)-dependent exocytotic glutamate release from rat cerebrocortical nerve terminals, but was ineffective against the Ca(2+)-independent glutamate release. The P2X7 competitive antagonist A-438079 eliminated both exocytotic and non-exocytotic P2X7 receptor-evoked glutamate release. Selective inhibition of exocytotic glutamate release indicates that calmidazolium inhibits events dependent on the function of native rat P2X7 receptors as Ca(2+) channels, and suggests that it can be used as a tool to dissociate P2X7-evoked exocytotic from non-exocytotic glutamate release.     

Transmitter Glutamate Release from Isolated Nerve Terminals: Evidence for Biphasic Release and Triggering by Localized Ca2+

The kinetics of Ca2(+)-dependent release of glutamate from guinea-pig cerebrocortical synaptosomes evoked by KCl or 4-aminopyridine are investigated using a continuous fluorimetric assay. Release by both agents is biphasic, with a rapid phase complete within 2 s followed by a more extensive slow phase with a half-maximal release in 52 s for KCl-evoked release and greater than 120 s for 4-aminopyridine-evoked release. The two phases of glutamate release may reflect a dual localization of releasable vesicles at the active zone and in the bulk cytoplasm. Decreasing depolarization depresses the extent rather than increasing the time for half-maximal Ca2(+)-dependent release. Both the fast and the slow phases of glutamate release require external Ca2+ and cytoplasmic ATP. KCl depolarization produces a transient "spike" of cytoplasmic free Ca2+ [( Ca2+]c), which recovers to a plateau; the major component of glutamate release occurs during this plateau. Predepolarization in the absence of added external Ca2+, to inhibit transient Ca2+ channels, does not affect the subsequent glutamate release evoked by Ca2+ readdition. Thus, release involves primarily noninactivating Ca2+ channels. For a given increase in [Ca2+]c, KCl and 4-aminopyridine cause equal release of glutamate, while ionomycin releases much less glutamate. This lowered efficiency is not due to ATP depletion. It is concluded that glutamate exocytosis is evoked by localized Ca2+ entering through noninactivating voltage-dependent Ca2+ channels and that nonlocalized Ca2+ entry with ionomycin is inefficient.     

Prostaglandin synthesis in rat brain astrocytes is under the control of the n-3 docosahexaenoic acid, released by group VIB calcium-independent phospholipase A2

In the current study, we reveal that in astrocytes the VIB Ca(2+)-independent phospholipase A(2) is the enzyme responsible for the release of docosahexaenoic acid (22:6n-3). After pharmacological inhibition and siRNA silencing of VIB Ca(2+)-independent phospholipase A(2), docosahexaenoic acid release was strongly suppressed in astrocytes, which were acutely stimulated (30 min) with ATP and glutamate or after prolonged (6 h) stimulation with the endotoxin lipopolysaccharide. Docosahexaenoic acid release proceeds simultaneously with arachidonic acid (20:4n-6) release and prostaglandin liberation from astrocytes. We found that prostaglandin production is negatively controlled by endogenous docosahexaenoic acid, since pharmacological inhibition and siRNA silencing of VIB Ca(2+)-independent phospholipase A(2) significantly amplified the prostaglandin release by astrocytes stimulated with ATP, glutamate, and lipopolysaccharide. Addition of exogenous docosahexaenoic acid inhibited prostaglandin synthesis, which suggests that the negative control of prostaglandin synthesis observed here is likely due to competitive inhibition of cyclooxygenase-1/2 by free docosahexaenoic acid. Additionally, treatment of astrocytes with docosahexaenoic acid leads to the reduction in cyclooxygenase-1 expression, which also contributes to reduced prostaglandin production observed in lipopolysaccharide-stimulated cells. Thus, we identify a regulatory mechanism important for the brain, in which docosahexaenoic acid released from astrocytes by VIB Ca(2+)-independent phospholipase A(2) negatively controls prostaglandin production.