Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K⁺ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba²⁺ could substitute for 1 m M Ca²⁺ in evoking the release of endogenous glutamate. In addition, Ba²⁺ was found to evoke glutamate release in the absence of K⁺-induced depolarization. Ba²⁺ (1–10 m M ) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [³H]-tetraphenylphosphonium cation distribution. Ba²⁺ partially inhibited the increase in synaptosomal K⁺ efflux produced by depolarization, as reflected by the redistribution of radiolabeled ⁸⁶Rb⁺. The release evoked by Ba²⁺ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca²⁺] increased during stimulation by approximately 200 n M , but cytosolic [Ba²⁺] increased by more than 1 μ M . Taken together, our results indicate that Ba²⁺ initially depolarizes synaptosomes most likely by blocking a K⁺ channel, which then activates TTX-sensitive Na⁺ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca²⁺channels to evoke neurotransmitter release directly. Though Ba²⁺-evoked glutamate release was comparable in level to that obtained with K⁺-induced depolarization in the presence of Ca²⁺, the apparent intrasynaptosomal level of Ba²⁺ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca²⁺.     

Excitatory Amino Acid Receptors and Depolarization-Induced Ca2+ Influx into Hippocampal Slices

Hippocampal brain slices were incubated with depolarizing agents or excitatory amino acids either alone or in the presence of excitatory amino acid antagonists [omega-phosphonic alpha-aminocarboxylic acids--2-amino-4-phosphonobutyric acid (AP4), 2-amino-5-phosphonovaleric acid (AP5), or 2-amino-7-phosphonoheptanoic acid (AP7)--or gamma-D-glutamylaminomethylsulphonic acid (GAMS)] or a calcium-channel blocker, (S)-1-(3-methoxyphenyl)-3-methylaza-7-cyano-7-(3,4-dimethoxyphenyl )-8-methyl- nonane hydrochloride [(-)-D888]. The uptake of 45Ca2+ and the efflux of glutamate or aspartate induced by veratrine or high K+ was blocked (54-76%) by AP7 (IC50 46-250 microM). AP5 and AP4 were less effective. (-)-D888 (10 microM) caused 100% block of evoked 45Ca2+ uptake. Uptake of 45Ca2+ induced by exogenous glutamate, aspartate, and N-methyl-D-aspartate (NMDA) was also inhibited by AP7, whereas GAMS completely blocked the action of kainate and partially blocked that of glutamate. The action of NMDA in stimulating 45Ca2+ uptake was Mg2+-sensitive, low Mg2+ levels in the incubation medium selectively enhancing the response. It is concluded that Ca2+ uptake evoked by excitatory amino acids is receptor-mediated, and that released excitatory amino acids are responsible for a large part of the action of veratrine and high K+ in stimulating 45Ca2+ uptake.     

12-Lipoxygenase Products Attenuate the Glutamate Release and Ca2+ Accumulation Evoked by Depolarization of Hippocampal Mossy Fiber Nerve Endings

Presynaptic correlates of evoked neurotransmitter release include a rise in cytosolic free calcium level and the calcium-dependent liberation of unesterified arachidonic acid. It has been proposed that lipoxygenase metabolites produced from arachidonic acid may constitute an endogenous feedback system for the modulation of neurotransmitter release. The results of the present study are in agreement with this hypothesis. It was demonstrated that membrane depolarization evoked the release of endogenous glutamate from hippocampal mossy fiber synaptosomes, as well as the accumulation of intraterminal free calcium. The presence of 12-lipoxygenase products attenuated both the induced release of glutamate and the increase in calcium content, whereas 5- or 15-lipoxygenase metabolites were ineffective. A role for lipoxygenase products in the negative modulation of mossy fiber secretion processes was further indicated by the observations that low concentrations of the lipoxygenase inhibitor nordihydroguaiaretic acid (0.1-10 microM) potentiated the glutamate release and calcium accumulation induced by membrane depolarization. Therefore, we suggest that 12-lipoxygenase metabolites provide a presynaptic inhibitory signal that limits neurotransmitter release from hippocampal mossy fiber terminals.     

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.     

Astroglial Gap Junction Communication Is Increased by Treatment with Either Glutamate or High K+ Concentration

Abstract: Astroglia are extensively coupled by gap junctions and form a functional syncytium. Astroglial gap junctions are thought to be involved in the spatial buffering of K⁺ in vivo and in the Ca²⁺ waves seen on glutamate receptor activation. The conductivity of gap junctions is regulated by several second messengers, with up-regulation by cyclic AMP and down-regulation through activation of protein kinase C, decreases in intracellular pH, or increases in the free cytosolic Ca²⁺ concentration. The results presented here indicate that dye coupling of astroglia is significantly up-regulated by membrane depolarization, both by increases in the extracellular K⁺ concentration and directly by ionophores. Furthermore, glutamate, kainate, and quisqualate, which depolarize astroglial cells through activation of ionotropic receptors, also increase dye coupling in astroglia. The effect of kainate and quisqualate was reversed by 6-cyano-7-nitroquinoxaline-2,3-dione, an inhibitor of the ionotropic glutamate receptor. A dose-dependent decrease in dye coupling was seen when the cells were injected with increasing concentrations of Ca²⁺. However, if the cells were simultaneously depolarized, the inhibitory effect of Ca²⁺ on gap junctional conductance was reversed. Significant increases over basal coupling was attained when the cells were injected with Ca²⁺ if they were treated with kainate or K⁺. These data suggest that ligands that depolarize astroglia enhance gap junction communication between astroglia and that this enhancement may be important in maintaining communication between astroglia in the face of elevated Ca²⁺ levels.     

Dantrolene Prevents Glutamate Cytotoxicity and Ca2+ Release from Intracellular Stores in Cultured Cerebral Cortical Neurons

Using primary cultures of cerebral cortical neurons, it has been demonstrated that the antihyperthermia drug dantrolene completely protects against glutamate-induced neurotoxicity. Furthermore, in the presence of extracellular calcium, dantrolene reduced the glutamate-induced increase in the intracellular calcium concentration by 70%. In the absence of extracellular calcium, this glutamate response was completely blocked by dantrolene. Dantrolene did not affect the kinetics of [3H]glutamate binding to membranes prepared from similar cultures. These results indicate that release of calcium from intracellular stores is essential for the propagation of glutamate-induced neuronal damage. Because it is likely that glutamate is involved in neuronal degeneration associated with ischemia and hypoxia, the present findings might suggest that dantrolene and possibly other drugs affecting intracellular calcium pools might be of therapeutic interest.     

Interaction Between A1 Adenosine and Class II Metabotropic Glutamate Receptors in the Regulation of Purine and Glutamate Release from Rat Hippocampal Slices

Abstract: Electrical stimulation of rat hippocampal slices evoked the release of excitatory amino acids and purines, as reflected by a time-dependent increase in the extracellular levels of glutamate and adenosine, as well as by the increased efflux of radioactivity in slices preloaded with both [¹⁴C]glutamate and [³H]adenosine. The evoked release of excitatory amino acids and purines was amplified when slices were exposed to 8-cyclopentyl-1,3-dipropylxanthine (a selective A1 adenosine receptor antagonist), (+)-α-methyl-4-carboxyphenylglycine [a mixed antagonist of metabotropic glutamate receptors (mGluRs)], or (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (a selective antagonist of class II mGluRs). In contrast, 2-chloro-N⁶-cyclopentyladenosine (CCPA; a selective A1 receptor agonist) or (2S,1R,2R,3R)-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; a selective agonist of class II mGluRs) reduced the evoked release of excitatory amino acids and purines. CCPA and DCG-IV also reduced the increase in cyclic AMP formation induced by either forskolin or electrical stimulation in hippocampal slices. The inhibitory effect of CCPA and DCG-IV on release or cyclic AMP formation was less than additive. We conclude that the evoked release of excitatory amino acids and purines is under an inhibitory control by A1 receptors and class II mGluRs, i.e., mGluR2 or 3, which appear to operate through a common transduction pathway. In addition, although these receptors are activated by endogenous adenosine and glutamate, they can still respond to pharmacological agonists. This provides a rationale for the use of A1 or class II mGluR agonists as neuroprotective agents in experimental models of excitotoxic neuronal degeneration.     

Electrical Stimulation of the Stratum Radiatum Increases the Release and Neosynthesis of Aspartate, Glutamate, and γ-Aminobutyric Acid in Rat Hippocampal Slices

The release of endogenous aspartic, glutamic, and gamma-aminobutyric acids (Asp, Glu, GABA, respectively) was measured in the effluent from superfused hippocampal slices using a new and sensitive mass spectrometric method. The stimulation of the stratum radiatum of the rat dorsal hippocampus caused a Ca2+-dependent increase in the release of these amino acids. This release was accompanied by an increase in the incorporation of [13C2] from [13C]glucose into Asp, Glu, and GABA, suggesting an increase in their neosynthesis. The removal of Ca2+ from the superfusion fluid brought about a marked decrease in Asp and Glu release at rest, and prevented their stimulation-evoked release and the appearance of population spikes. The results support the hypothesis that Asp and Glu are excitatory neurotransmitters in intrinsic hippocampal circuits and are possibly released from the Schaffer collaterals and commissural fibres. The increase in GABA release and neosynthesis during stimulation of the stratum radiatum could be related to recurrent inhibition evoked by transsynaptic stimulation of the pyramidal cells.     

In Vivo Release from Cerebral Cortex of [14C]Glutamate Synthesized from [U-14C]Glutamine

Awake, unrestrained, and behaviourally normal animals with superfusion cannulae implanted over the sensorimotor cortex were used in a study of the capacity of infused [U-14C]glutamine for labelling glutamate and other amino acids released by depolarising stimuli. A spontaneous background release of [14C]glutamate was detected. This was increased by tityustoxin (1 microM). The specific radioactivity of glutamate increased eightfold during the evoked-release period. [14C]Aspartate was also detected and showed increased release, but not increased specific labelling, in response to depolarisation. Evoked gamma-aminobutyric acid (GABA) release occurred but only small amounts of [14C]GABA were detected. Glutamine showed increased rates of uptake to the sensorimotor cortex during stimulation periods, suggesting an accelerated breakdown via glutaminase.     

Ca2+ -Dependent Release from Rat Brain of Cannabinoid Receptor Binding Activity

As a result of the identification, pharmacological characterization, and localization of the cannabinoid receptor in the CNS, the existence of an endogenous ligand for this receptor can be hypothesized. Following the premise that such a substance could have the properties of a neuromodulator being stored in intracellular vesicles, we tested the ability of increased intracellular Ca2+ levels to stimulate release. We demonstrate here that the Ca2+ ionophore A23187 can induce release of cannabinoid-like binding activity in the presence but not in the absence of Ca2+. The effect of A23187 was maximal at 1.2 microM, consistent with vesicular release. It was necessary to increase the concentration of extracellular free Ca2+ to greater than 60 nM to evoke release. The released cannabinoid-like binding activity displaced [3H]CP-55940 binding to cannabinoid receptors in rat synaptosomal membranes in a concentration-dependent manner. This is the first report of a substance present endogenously in brain that can be released in a Ca(2+)-dependent manner and that binds to the cannabinoid receptor.     

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.     

Reductions of Γ-Aminobutyric Acid and Glutamate Uptake and (Na++ K+)-ATPase Activity in Brain Slices and Synaptosomes by Arachidonic Acid

Arachidonic acid, a major polyunsaturated fatty acid of membrane phospholipids in the CNS, reduced the high-affinity uptake of glutamate and gamma-aminobutyric acid (GABA) in both rat brain cortical slices and synaptosomes. alpha-Aminoisobutyric acid uptake was not affected. Intrasynaptosomal sodium was increased concomitant with decreased (Na+ + K+)-ATPase activity in synaptosomal membranes. The reduction of GABA uptake in synaptosomes could be partially reversed by alpha-tocopherol. The inhibition of membrane-bound (Na+ + K+)-ATPase by arachidonic acid was not due to a simple detergent-like action on membranes, since sodium dodecyl sulfate stimulated the sodium pump activity in synaptosomes. These data indicate that arachidonic acid selectively modifies membrane stability and integrity associated with reductions of GABA and glutamate uptake and of (Na+ + K+)-ATPase activity.     

Influence of Na+, K+, and Ca2+ on Glutamine Synthesis and Distribution in Rat Brain Cortex Slices: A Possible Linkage of Glutamine Synthetase with Cerebral Transport Processes and Energetics in the Astrocytes

The ouabain-induced suppression of glutamine synthesis and retention in incubating rat brain cortex slices was found to be mimicked by changes in the cationic content of the incubation medium, which cause an increase in the intracellular [Na+] and a decrease in the intracellular [K+]. The suppression of glutamine synthesis (and fixation of ammonia) was also found to take place when Ca2+ was omitted from the incubation medium. This occurred whether endogenous or exogenous glutamate was the substrate for glutamine synthesis. The suppressions cannot be due solely to an effect on glutamate uptake, because the uptake is not markedly affected by these conditions. The results show that Na+, K+, and Ca2+ influence the synthesis and distribution of glutamine in the brain. They suggest that Ca2+ and the Na+, K+ pump may serve a role in regulating the activity of ATP-dependent glutamine synthetase, a key enzyme of the glutamate-glutamine cycle, located in the astrocytes. This may be mediated via a direct effect on the enzyme or through an effect on the production of ATP.     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.     

Effect of Age on K+-Induced Cytosolic Ca2+ Changes in Rat Cortical Synaptosomes

45Ca2+ uptake and cytosolic Ca2+ concentrations [( Ca2+]i) were measured in synaptosomes prepared from the cerebral cortex of 3-, 16-, and 24-month-old male Charles River Wistar rats. Electron-microscopic examination demonstrated no morphological differences between the synaptosomes prepared from 3- and 24-month-old rats. The fast phase of Ca2+ uptake was reduced in the 24-month-old animals as compared to the 3-month-old ones (-23%, p less than 0.001), whereas no difference was found between the 16- and the 3-month-old rats. Age did not modify [Ca2+]i, as measured by the quin 2 technique, both at rest and immediately after depolarization with 50 mM K+. The Ca2+ load following depolarization was cleared in about 13 min in the 3-month-old rats. The rate of clearance was significantly slower both in the 16- (p less than 0.01) and in the 24-month-old rats (p less than 0.0001). The addition of verapamil (60 microM) after depolarization restored [Ca2+]i to resting level in aged rats at the same rate as in young rats. A prolonged Ca2+ influx, therefore, may be responsible for the slower clearance of Ca2+ load in aged rats.     

Ca2+ dependence and La3+ interference of ultraviolet radiationinduced K+ efflux from rose cells

A low fluence of ultraviolet radiation (UV) causes cultured cells of Rosa damascena Mill cv. Gloire de Guilan to lose intracellular K⁺. This effect required the presence of Ca²⁺ in the medium. A reduction in the concentration of free Ca²⁺ to 10⁻⁵ M with ethyleneglycol-bis-(β-aminoethyl-ether)-N.N.N',N'-tetraacetic acid (EGTA) buffer inhibited the UV-stimulated efflux; this was correlated with a discharge of the membrane potential and a stimulation of the leakage of K⁺ from unirradiated cells. All the same effects were seen with La³⁺ at 0.2 m M. At 0.02 m M La³⁺, the UV-stimulated efflux of K⁺ was blocked without concomitant effects on the membrane potential or K⁺ efflux from control cells. It is suggested that removal of Ca²⁺ blocks or masks the UV-induced leakage of K⁺ by destabilizing the plasma membrane. In addition, La³⁺ may specifically inhibit the UV-stimulated opening of K⁺ or anion channels.     

Release of Neuropeptide FF, an Anti-Opioid Peptide, in Rat Spinal Cord Slices Is Voltage- and Ca2+-Sensitive: Possible Involvement of P-Type Ca2+ Channels

Abstract: Neuropeptide FF (NPFF), an FMRFamide-like peptide with antiopioid properties, inhibits morphine-induced analgesia but also produces hyperalgesia. In the present study, the mechanisms of NPFF release were investigated in an in vitro superfusion system with rat spinal cord slices. The opening of voltage-sensitive Na⁺ channels with veratridine (20 µ M ) induced calcium-dependent NPFF release, which was abolished by tetrodotoxin (1 µ M ), suggesting that NPFF release depends on nerve impulse activity. We also showed that NPFF release was a function of the extent of depolarization and was calcium dependent. The 30 m M K⁺-induced release was blocked by Co²⁺ or Ni²⁺ (2.5 m M ) but was unaffected by Ca²⁺ channel blockers of the L type—Cd²⁺ (100 µ M ), nifedipine or nimodipine (10 µ M ), diltiazem (20 µ M ), or verapamil (50 µ M )—or the N type—ω-conotoxin GVIA (1 µ M ). In contrast, ω-agatoxin IVA (1 µ M ) led to a 65% reduction in NPFF release, suggesting that P-type Ca²⁺ channels play a prominent role. The 35% remaining release resulted from activation of an unknown subtype. The NPFF-like material in superfusates recognized spinal NPFF receptors, suggesting that NPFF release in the spinal cord has a physiological role.     

Adenosine Receptor Agonists Inhibit K+-Evoked Ca2+ Uptake by Rat Brain Cortical Synaptosomes

Abstract: The uptake of Ca²⁺ by a K⁺-depolarized rat brain cerebral cortical crude synaptosomal preparation (P₂ fraction) was investigated. The characteristics of the Ca²⁺ uptake system are similar to those observed by other investigators. The preparation is also a suitable model with which to study the effects of adenosine on Ca²⁺ uptake and neurotransmitter release, as it is generally accepted that K⁺-evoked Ca²⁺ uptake is intimately related to depolarization-induced release of neurotransmitters. We have demonstrated that an extracellular receptor is involved in mediating the adenosine-evoked inhibition of K⁺-evoked Ca²⁺ uptake. The pharmacological properties of the receptor suggest that it may be similar in some respects to the A₂-receptor associated with adenylate cyclase. The adenosine uptake inhibitor, dipyridamole, potentiated the action of adenosine, suggesting that re-uptake is important in controlling the extracellular adenosine concentration and thus in the regulation of the adenosine receptor. The adenosine receptor antagonist theophylline inhibited the effects of adenosine. Calmodulin inhibited K⁺- evoked uptake of Ca²⁺ by the synaptosomal fraction.     

Nitric Oxide Disrupts Ca2+ Homeostasis in Hippocampal Neurons

Abstract: Nitric oxide has been recognized in recent years as an important mediator of neuronal toxicity, which in many cases involves alterations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In [Ca²⁺]_i fluorimetric experiments on cultured hippocampal neurons, the nitric oxide-releasing agent S -nitrosocysteine produced a delayed rise in [Ca²⁺]_i over a 20-min exposure, which was accompanied by a progressive slowing of the kinetics of recovery from depolarization-induced [Ca²⁺]_i transients. These effects were blocked by oxyhemoglobin and by superoxide dismutase, confirming nitric oxide as the responsible agent, and suggesting that they involved peroxynitrite formation. Similar alterations of [Ca²⁺]_i homeostasis were produced by the mitochondrial ATP synthase inhibitor oligomycin, and when an ATP-regenerating system was supplied via the patch pipette in combined whole-cell patch-clamp-[Ca²⁺]_i fluorimetry experiments, S -nitrosocysteine had no effect on the resting [Ca²⁺]_i or on the recovery kinetics of [Ca²⁺]_i transients induced by direct depolarization. We conclude that prolonged exposure to nitric oxide disrupts [Ca²⁺]_i homeostasis in hippocampal neurons by impairing Ca²⁺ removal from the cytoplasm, possibly as a result of ATP depletion. The resulting persistent alterations in [Ca²⁺]_i may contribute to the delayed neurotoxicity of nitric oxide.     

N-Methyl-D-Aspartate Receptor Activation and Ca2+ Account for Poor Pyramidal Cell Structure in Hippocampal Slices

The CA1 pyramidal cells appear damaged in micrographs of guinea pig hippocampal slices incubated in normal physiological buffer at 36–37°C. This is remedied if slices are incubated in modified buffers for the first 45 min. Cell morphology is improved if this buffer is devoid of added Ca²⁺ and much improved if it contains N-methyl-D-aspartate (NMDA) receptor antagonists or 0 mM Ca²⁺ and 10 mM Mg²⁺. The cells then appear similar to CA1 pyramidal cells in situ. These findings support the notion that NMDA receptor activation and Ca²⁺, acting in the period immediately after slice preparation, permanently damage CA1 pyramidal cells in vitro.