Mechanisms of [(3)H]glycine release from mouse spinal cord synaptosomes selectively labeled through GLYT2 transporters

Glycine release has been rarely studied. The aim of this work was to characterize the release of the amino acid from spinal cord glycinergic nerve endings selectively pre-labeled through glycine transporters of the GLYT2 type. Purified mouse spinal cord synaptosomes were incubated with [(3)H]glycine in the presence of the GLYT1 blocker N-[(3R)-3-([1,1'-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine hydrochloride and exposed in superfusion to varying concentrations of KCl, 4-aminopyridine (4-AP), or veratridine. KCl (< or = 15 micromol/L), 4-AP (up to 1 mmol/L), and veratridine (< or = 0.3 micromol/L)-provoked [(3)H]glycine release by external Ca2+-dependent, botulinum toxin C(1)-sensitive, exocytosis. The overflows evoked by higher concentrations of K+ or veratridine involved external Ca2+-independent mechanisms of different nature. Only the overflow evoked by 3 or 10 micromol/L veratridine occurred totally (3 micromol/L) or in part (10 micromol/L) by transporter reversal, being sensitive to the GLYT2 blockers 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminociclopentyl)-methyl] benzamide or O-[(2-benzyloxyphenyl-3-flurophenyl)methyl]-l-serine; in contrast, the external Ca2+-independent [(3)H]glycine overflow provoked by 50 mmol/L K+ was transporter-independent. This component of K+-evoked overflow and the GLYT2-independent portion of the 10 micromol/L veratridine-evoked overflow, were largely sensitive to the vesicle depletor bafilomycin or BAPTA-AM and were prevented by blocking the mitochondrial Na+/Ca2+ exchanger with 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one, indicating the involvement of exocytosis triggered by intraterminal mitochondrial Ca2+ ions.     

Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 micromol/L) increased endogenous glutamate or [3H]D-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.     

Glycine release provoked by disturbed Na⁺, Na⁺ and Ca²⁺ homeostasis in cerebellar nerve endings: roles of Ca²⁺ channels, Na⁺/Ca²⁺ exchangers and GlyT2 transporter reversal

Glycine release provoked by ion dysregulations typical of some neuropathological conditions was analyzed in cerebellar synaptosomes selectively pre-labelled with [3H]glycine through GlyT2 transporters and exposed in superfusion to KCl, 4-aminopyridine (4-AP) or veratridine. The overflows caused by relatively low concentrations of the releasers were largely external Ca2?-dependent. Higher concentrations of KCl (50 mM) or veratridine (10 μM), but not of 4-AP (1 mM), involved also external Ca2?-independent mechanisms. GlyT1-mediated release could not be observed; only the external Ca2?-independent veratridine-evoked overflow occurred significantly by GlyT2 reversal. None of the three depolarizing agents activated store-operated or transient receptor potential or L-type Ca2? channels. The overflows caused by KCl or 4-AP occurred in part by N- and P/Q-type voltage-sensitive calcium channel-dependent exocytosis. Significant portions of the external Ca2?-dependent overflow evoked by KCl or 4-AP (and all that caused by veratridine) were mediated by reverse plasmalemmal Na?/Ca2? exchangers. Significant contribution to the overflows evoked by KCl or veratridine came from Ca2? originated through mitochondrial Na?/Ca2? exchangers. Ca2?-induced Ca2? release (CICR) mediated by inositoltrisphosphate receptors (InsP?Rs) represents the final trigger of the glycine release evoked by high KCl. The overflows evoked by 4-AP or, less so, by veratridine also involved InsP?R-mediated CICR and, in part, CICR mediated by ryanodine receptors. To conclude, ionic dysregulations typical of ischemia and epilepsy caused glycine release in cerebellum by multiple differential mechanisms that may represent potential therapeutic targets.     

Multiple mechanisms of transmitter release evoked by "pathologically" elevated extracellular [K+]: involvement of transporter reversal and mitochondrial calcium

The release of [3H]GABA evoked by depolarization with various concentrations of KCl was studied using superfused rat cerebrocortex synaptosomes. Elevating [K+] produced release of [3H]GABA over basal which was increasingly less dependent on external Ca2+ but more sensitive to the GABA transporter blocker SKF 100330 A. Accordingly, the sensitivity to clostridial toxins of the depolarization-evoked amino acid release was inversely correlated to the concentration of KCl used. However, at 50 mM K+, one-third of the stimulated release remained which was external Ca2+-independent but insensitive to SKF 100330 A. This release was prevented by BAPTA, thapsigargin or dantrolene; it also was inhibited by blocking in mitochondria the ATP production with oligomycin, the H+-dependent Ca2+ uniporter with RU 360, the Na+/Ca2+ exchanger with CGP 37157 or by lowering extraterminal [Na+]. In fluorescence experiments with fura-2/AM, 50 mM K+ (in Ca2+ free medium) caused elevation of cytosolic [Ca2+] that was sensitive to thapsigargin or CGP 37157; these compounds produced partially additive effects. When exocytosis was monitored with the fluorescent dye acridine orange, the fluorescence elicited by 50 mM K+ was sensitive to thapsigargin or CGP 37157, which produced additive effects, and to low-Na+ media. To conclude, extracellular K+ concentrations occurring in the CNS in certain pathological conditions provoke GABA release by mechanisms different from classical exocytosis. These include carrier-mediated release and internal Ca2+-dependent exocytosis; in the latter, mitochondrial Ca2+ seems to play a primary role.     

Glycinergic nerve endings in hippocampus and spinal cord release glycine by different mechanisms in response to identical depolarizing stimuli

Studies on hippocampal glycine release are extremely rare. We here investigated release from mouse hippocampus glycinergic terminals selectively pre-labelled with [³H]glycine through transporters of the GLYT2 type. Purified synaptosomes were incubated with [³H]glycine in the presence of the GLYT1 blocker NFPS to abolish uptake (∼ 30%) through GLYT1. The non-GLYT1-mediated uptake was entirely sensitive to the GLYT2 blocker Org25543. Depolarization during superfusion with high-K⁺ (15–50 mmol/L) provoked overflows totally dependent on external Ca²⁺, whereas in the spinal cord the 35 or 50 mmol/L KCl-evoked overflow (higher than that in hippocampus) was only partly dependent on extraterminal Ca²⁺. In the hippocampus, the Ca²⁺-dependent 4-aminopyridine (1 mmol/L)-evoked overflow was five-fold lower than that in spinal cord. The component of the 10 μmol/L veratridine-induced overflow dependent on external Ca²⁺ was higher in the hippocampus than that in spinal cord, although the total overflow in the hippocampus was only half of that in the spinal cord. Part of the veratridine-evoked hippocampal overflow occurred by GLYT2 reversal and part by bafilomycin A₁-sensitive exocytosis dependent on cytosolic Ca²⁺ generated through the mitochondrial Na⁺/Ca²⁺ exchanger. As glycine sites on NMDA receptors are normally not saturated, understanding mechanisms of glycine release should facilitate pharmacological modulation of NMDA receptor function.     

The sensitivity of catecholamine release to botulinum toxin C1 and E suggests selective targeting of vesicles set into the readily releasable pool

The impact of syntaxin and SNAP-25 cleavage on [3H]noradrenaline ([3H]NA) and [3H]dopamine ([3H]DA) exocytotic release evoked by different stimuli was studied in superfused rat synaptosomes. The external Ca2+-dependent K+-induced [3H]catecholamine overflows were almost totally abolished by botulinum toxin C1 (BoNT/C1), which hydrolyses syntaxin and SNAP-25, or by botulinum toxin E (BoNT/E), selective for SNAP-25. BoNT/C1 cleaved 25% of total syntaxin and 40% of SNAP-25; BoNT/E cleaved 40% of SNAP-25 but left syntaxin intact. The GABA uptake-induced releases of [3H]NA and [3H]DA were differentially affected: both toxins blocked the former, dependent on external Ca2+, but not the latter, internal Ca2+-dependent. BoNT/C1 or BoNT/E only slightly reduced the ionomycin-evoked [3H]catecholamine release. More precisely, [3H]NA exocytosis induced by ionomycin was sensitive to toxins in the early phase of release but not later. The Ca2+-independent [3H]NA exocytosis evoked by hypertonic sucrose, thought to release from the readily releasable pool (RRP) of vesicles, was significantly reduced by BoNT/C1. Pre-treating synaptosomes with phorbol-12-myristate-13-acetate, to increase the RRP, enhanced the sensitivity to BoNT/C1 of [3H]NA release elicited by sucrose or ionomycin. Accordingly, cleavage of syntaxin was augmented by the phorbol-ester. To conclude, our results suggest that clostridial toxins selectively target exocytosis involving vesicles set into the RRP.     

Involvement of KCNQ2 subunits in [3H]dopamine release triggered by depolarization and pre-synaptic muscarinic receptor activation from rat striatal synaptosomes

KCNQ2 and KCNQ3 subunits encode for the muscarinic-regulated current (I(KM)), a sub-threshold voltage-dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of I(KM) in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the I(KM) activator retigabine (0.01-30 micromol/L; Emax = 54.80 +/- 3.85%; IC50 = 0.50 +/- 0.36 micromol/L). The I(KM) blockers tetraethylammonium (0.1-3 mmol/L) and XE-991 (0.1-30 micromol/L) enhanced K+-evoked [3H]DA release and prevented retigabine-induced inhibition of depolarization-evoked [3H]DA release. Retigabine-induced inhibition of K+-evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti-KCNQ2 polyclonal antibodies, an effect prevented by antibody pre-absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1-300 micromol/L) potentiated 9 mmol/L [K+]e-evoked [3H]DA release (Emax = 155 +/- 9.50%; EC50 = 25 +/- 1.80 micromol/L). OXO (100 micromol/L)-induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1-10 nmol/L) and abolished by the M3-preferring antagonist 4-diphenylacetoxy N-methylpiperidine methiodide (1 micromol/L), but was unaffected by the M1-selective antagonist MT-7 (10-100 nmol/L) or by Pertussis toxin (1.5-3 microg/mL), which uncouples M2- and M4-mediated responses. Finally, OXO-induced potentiation of depolarization-induced [3H]DA release was not additive to that produced by XE-991 (10 micromol/L), was unaffected by retigabine (10 micromol/L), and was abolished by synaptosomal entrapment of anti-KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, I(KM) channels containing KCNQ2 subunits regulate depolarization-induced DA release and that I(KM) suppression is involved in the reinforcement of depolarization-induced DA release triggered by the activation of pre-synaptic muscarinic heteroreceptors.     

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)     

Rapid Release of [3H]Dopamine from Median Eminence and Striatal Synaptosomes

Release of preaccumulated, tritium-labeled dopamine ([3H]DA) from preparations of isolated nerve terminals (synaptosomes) of rat median eminence (ME) and corpus striatum (CS) was examined over short time intervals (1-20 s). In both preparations, basal efflux of [3H]DA was linear with time. Depolarization with high K+ resulted in an initial rapid release of [3H]DA which stabilized by 20 s, whereas veratridine elicited an increased rate of release over basal levels that was linear over the first 20 s. The calculated rate constants of release for both the initial phase of K+- and the veratridine-stimulated release were approximately threefold greater in CS than in ME synaptosomes. The major component of the high K+-induced release of [3H]DA from both synaptosome preparations increased as a graded function of [Ca2+]o. However, a smaller component, independent of external Ca2+, existed in both ME and CS synaptosomes. Increasing the [Mg2+] in the external solution resulted in a right shift of both the [K+]o and the [Ca2+]o dose-response curves, consistent with actions of Mg2+ on screening surface membrane charges and blocking voltage-dependent Ca2+ channels. In all studies, steady-state uptake of the [3H]DA was about twofold greater into CS than into ME synaptosomes. Moreover, the fraction of incorporated [3H]DA released by stimulation from the CS was much greater than that released from ME synaptosomes. These data are consistent with differences between these two types of dopaminergic terminals with respect to packaging and/or distribution of the accumulated neurotransmitter in intraneuronal pools, as well as marked differences in the apparent kinetics of DA release.     

Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools

The aim of the present paper was to determine whether the release of glutamate from putative "glutamergic" terminals in the cerebellum is influenced by gamma-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent away; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors. GABA and muscimol at low concentrations (2--20 micrometers) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate "reuptake pool") from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of "glutamergic" nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.     

Characterization of depolarization-coupled release of glutamate from cultured mouse cerebellar granule cells using DL-threo-beta-benzyloxyaspartate (DL-TBOA) to distinguish between the vesicular and cytoplasmic pools

Release of preloaded [3H]D-aspartate in response to depolarization induced by N-methyl-D-aspartate (NMDA) or the endogenous agonist glutamate was characterized using cultured glutamatergic cerebellar granule neurons. Release from the vesicular and the cytoplasmic glutamate pools, respectively, was distinguished employing the competitive, non-transportable glutamate transport inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA). NMDA (300 microM)-induced release was enhanced (50%) by a simultaneous elevation of the extracellular potassium concentration to 15 mM, which lifts the voltage-dependent magnesium block of the NMDA receptors. This NMDA/K(+)-induced release was not sensitive to DL-TBOA (100 microM) but was inhibited by 75% in the presence of the unspecific calcium channel antagonist La(3+) (100 microM). Glutamate (100 microM) induced a large fractional release of the preloaded [3H]D-aspartate and in the presence of DL-TBOA the release was reduced by approximately 50%. In contrast, release evoked by 25 microM glutamate was not inhibited by DL-TBOA. These results indicate that the release elicited by 100 microM glutamate is comprised of a significant glutamate transporter-mediated component in addition to the vesicular release while the NMDA/K(+)-induced release is vesicular in nature. It is likely that the high glutamate concentration (100 microM) may facilitate heteroexchange of the preloaded [3H]D-aspartate.     

Direct evidence that release-stimulating alpha7* nicotinic cholinergic receptors are localized on human and rat brain glutamatergic axon terminals

The existence on glutamatergic nerve endings of nicotinic acetylcholine receptors (nAChRs) mediating enhancement of glutamate release has often been suggested but not demonstrated directly. Here, we study the effects of nAChR agonists on [3 H]-d-aspartate ([3 H]-d-ASP) release from synaptosomes superfused in conditions known to prevent indirect effects. Nicotinic receptor agonists, while unable to modify the basal [3 H]-d-ASP release from human neocortex or rat striatal synaptosomes, enhanced the Ca2+ -dependent exocytotic release evoked by K+ (12 mm) depolarization. Their rank order of potency were anatoxin-a > epibatidine > nicotine > ACh (+ atropine). The anatoxin-a effect, both in human and rat synaptosomes, was antagonized by mecamylamine, alpha-bungarotoxin or methyllycaconitine. The basal release of [3 H]ACh from human cortical synaptosomes was increased by (-)-nicotine (EC50 = 1.16 +/- 0.33 microm) or by ACh plus atropine (EC50 = 2.0 +/- 0.04 microm). The effect of ACh plus atropine was insensitive to alpha-bungarotoxin, methyllycaconitine or alpha-conotoxin MII, whereas it was totally antagonized by mecamylamine or dihydro-beta-erythroidine. To conclude, glutamatergic axon terminals in human neocortex and in rat striatum possess alpha7* nicotinic heteroreceptors mediating enhancement of glutamate release. Release-enhancing cholinergic autoreceptors in human neocortex are nAChRs with a pharmacological profile compatible with the alpha4beta2 subunit combination.     

Release of Endogenous and Newly Synthesized Glutamate and of Other Amino Acids Induced by Non-N-Methyl-D-Aspartate Receptor Activation in Cerebellar Granule Cell Cultures

Amino acid release studies were performed by an HPLC procedure using differentiated rat cerebellar granule cell cultures. Kainic acid (KA; 50 microM) caused an increase (about threefold) in the release of endogenous glutamate and a lesser, but statistically significant, increase in the release of glutamine, glycine, threonine, taurine, and alanine. Quisqualic acid (QA) and, to a lesser degree, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (both 50 microM) enhanced the release of the following amino acids in the order glutamate greater than aspartate greater than or equal to taurine, whereas the release of other amino acids was either unaffected or affected in a statistically nonsignificant way. The release of glutamate induced by KA was partially (43%) Ca2+ dependent. The other release-inducing effects of KA and QA were not Ca2+ dependent. In all cases, the evoked release could be prevented by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-2,3-hydroxy-7-nitroquinoxaline, and thus appeared to be receptor mediated. NMDA (5 and 50 microM) had no release-inducing activity. The KA-, QA-, and AMPA-evoked release of newly synthesized [3H]glutamate and [3H]aspartate (formed in the cells exposed to [3H]glutamine) was very similar to the evoked release of endogenous glutamate and aspartate. On the other hand, the release of preloaded D-[3H]aspartate (purified by HPLC in the various fractions analyzed, before radioactivity determination) induced by 50 microM KA was twice as high as that of endogenous glutamate. In the case of high [K+] depolarization, in contrast, the release of preloaded D-[3H]aspartate was approximately 30% lower than that of endogenous glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of electrically evoked [3H]-D-aspartate release from hippocampal slices

Electrical stimulation has certain advantages over chemical stimulation methods for the study of neurotransmitter release in brain slices. However, measuring detectable quantities of electrically evoked release of endogenous or radiolabeled markers of excitatory amino acid neurotransmitters has required current intensities or frequencies much higher than those usually required to study other transmitter systems. We demonstrate here that [3H]-D-aspartate (D-ASP) release can be detected from hippocampal slices at lower stimulation intensities in the presence of a glutamate reuptake inhibitor. Subsequently, we optimized the electrical stimulus parameters for characterizing electrically evoked D-ASP release. Under the experimental conditions described, greater than 90% of electrically evoked D-ASP release is calcium-dependent. Evoked D-ASP release is markedly reduced by pre-treating slices with the synaptic vesicle toxin bafilomycin A1 (BAF A1) or in the presence of 10-mM magnesium. Evoked D-ASP release is also reduced to variable degrees by N- and P/Q type voltage-sensitive calcium channel antagonists. Neither spontaneous efflux nor evoked D-ASP release were affected by NMDA, AMPA or group I metabotropic glutamate receptor (mGluR) antagonists. Evoked D-ASP release was reduced in the presence of an adenosine A1 receptor agonist and potentiated by treatment with a group I mGluR5 agonist. Evoked [3H]-D-ASP release was similar in magnitude to evoked [3H]-L-glutamate (L-GLU) release. Finally, in separate experiments using the same electrical stimulus parameters, more than 90% of electrically evoked endogenous L-GLU release was calcium dependent, a pattern similar to that observed for evoked [3H]-D-ASP release. Taken together, these results indicate that electrically evoked [3H]-D-ASP release mimics evoked glutamate release in brain slices under the experimental conditions employed in these studies.     

Effect of Peroxides on [3H]d-Aspartate Release from Bovine Isolated Retinae

In the present study, we investigated the effect of naturally occurring and synthetic peroxides on K+-depolarization-evoked release of [3H]D-aspartate from bovine isolated retinae. Furthermore, effect of peroxides on endogenous glutamate concentrations were measured by HPLC in bovine neural retinae and vitreous humor of eyes treated with hydrogen peroxide (H2O2) ex vivo. Both naturally occurring H2O2 (1-100 microM) and synthetic (cumene hydroperoxide, cuOOH; 1-100 microM) peroxides caused a concentration-dependent inhibition of K+-evoked [3H]D-aspartate release without affecting basal tritium efflux. The antioxidant, trolox (2 mM) prevented the inhibition of evoked [3H]D-aspartate overflow elicited by both H2O2 (30 microM) and cuOOH (10 microM). Inhibition of catalase by 3-amino-triazole (3- AT 100 mM) enhanced an inhibitory effect of a low concentration of H2O2 (1 microM) but antagonized the effect of H2O2 (30 microM) on K+-induced [3H]D-aspartate release. In ex vivo experiments, exogenously applied H2O2 (1-100 microM) also caused a concentration-related decrease in glutamate levels in the bovine retina. We conclude that peroxides can inhibit K+-evoked release of [3H]D-aspartate and also decrease endogenous glutamate concentrations in the bovine retina.     

Entrapping of impermeant probes of different size into nonpermeabilized synaptosomes as a method to study presynaptic mechanisms

Small molecules present during brain tissue homogenization are known to be entrapped within subsequently isolated synaptosomes. We have revisited this technique in view of its systematic utilization to incorporate into nerve endings impermeant probes of large size. Rat neocortical synaptosomes were prepared in the absence or in the presence of each of the following compounds: 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), tetanus toxin (TeTx) or its light chain (TeTx-LC), pertussis toxin (PTx), anti-syntaxin, or anti-SNAP25 monoclonal antibodies. Release of endogenous GABA and glutamate was then evoked by high K+ depolarization. GABA and glutamate overflows were inhibited by entrapped BAPTA and in synaptosomes prepared by homogenization in the presence of varying concentrations of TeTx or TeTx-LC. When synaptobrevin cleavage in synaptosomes entrapped with TeTx was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by western blotting, the extent of proteolysis was found to correspond quantitatively to that of release inhibition. GABA and glutamate overflows were increased by entrapped PTx; moreover, (-)-baclofen inhibited amino acid overflow more potently in standard than in PTx-containing synaptosomes. The overflows of GABA and glutamate were similarly decreased following incorporation of anti-syntaxin or anti-SNAP25 antibodies. Synaptosomal entrapping may be routinely used to internalize membrane-impermeant agents of different size in studies of presynaptic mechanisms.     

Evidence that alpha7 nicotinic receptor modulates glutamate release from mouse neocortical gliosomes

The presence of nicotinic receptors on astrocytes in human and rat brain has been previously demonstrated however their possible functional role is still poorly understood. In this study we investigated on the presence of nicotinic receptors on gliosomes, purified from mouse cortex, and on their role in eliciting glutamate release. Epibatidine significantly increased basal release of [3H]D-aspartate and of endogenous glutamate from mouse gliosomes but not from synaptosomes. This effect was prevented by methyllycaconitine, alpha-bungarotoxin and mecamylamine but not by dihydro-beta-erythroidine. Epibatidine provoked also a significant increase of calcium concentration in gliosomes but not in synaptosomes; the increase in [Ca2+]i induced by epibatidine and KCl in gliosomes was very similar to each other. The present results indicate that alpha7 nicotinic receptors exist on mouse cortical glial particles and stimulate glutamate release.     

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.     

Role of Astrocytes in Depolarization-Coupled Release of Glutamate in Cerebellar Cultures

Release of preloaded D-[3H]aspartate in response to depolarization induced by high potassium, N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) or the endogenous agonist glutamate was studied using cultured glutamatergic cerebellar granule neurons, cerebellar astrocytes, and corresponding cocultures. Release from the vesicular and the cytoplasmic glutamate pools, respectively, was distinguished employing the competitive, non-transportable glutamate transport inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA). The results indicate that the release in response to AMPA (30 microM) in the presence of cyclothiazide (50 microM) to block desensitization, was of a vesicular origin. Pulses of 55 mM K+ caused a DL-TBOA resistant efflux of preloaded D-[3H]aspartate from astrocytes, indicating that this release was not mediated by glutamate transporters. The results furthermore support the notion of an important function of the astrocytes in the uptake of released glutamate, because DL-TBOA caused a large, apparent increase in the depolarization-coupled release of preloaded D-[3H]aspartate in the cocultures, compared to neuronal monocultures.     

Characterization of the Inhibitory Action of Botulinum Neurotoxin Type A on the Release of Several Transmitters from Rat Cerebrocortical Synaptosomes

Under optimised conditions for intoxication, botulinum neurotoxin type A was shown to inhibit approximately 90% of Ca2+-dependent K+-evoked release of [3H]acetylcholine, [3H]noradrenaline, and [3H]dopamine from rat cerebrocortical synaptosomes; cholinergic terminals were most susceptible. In each case, the dose-response curve for the neurotoxin was extended, with about 50% of evoked release being inhibited at approximately 10 nM whereas 200 nM was required for the maximal blockade. This may suggest some heterogeneity in the release process. The action of the toxin was time and temperature dependent and appeared to involve binding and sequestration steps prior to blockade of release. The neurotoxin failed to exert any effect on synaptosomal integrity or on Ca2+-independent release of the transmitters tested; it produced only minimal changes in neurotransmitter uptake although small secondary effects were detected with cholinergic terminals. Blockade by the neurotoxin of Ca2+-dependent resting release of transmitter was apparent; Sr2+, Ba2+, or high concentrations of Ca2+ restored the resting release of 3H-catecholamine but not [3H]acetylcholine. Interestingly, none of the latter conditions or 4-aminopyridine could reverse the toxin-induced blockade of evoked release. This lack of specificity in its action on synaptosomes, and other published findings, lead to the conclusion that toxin-sensitive component(s) exist in all nerve terminals that are concerned with transmitter release.