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.     

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.     

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.     

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.     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Na+ entry via glutamate transporter activates the reverse Na+/Ca2+ exchange and triggers Ca(i)2+-induced Ca2+ release in rat cerebellar Type-1 astrocytes

We have previously demonstrated that rat cerebellar Type-1 astrocytes express a very active genistein sensitive Na(+)/Ca(2+) exchanger, which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of Cai(2+) with Fluo-3 demonstrate that the Cai(2+) signals associated long (> 20 s) periods of reverse operation of the Na(+)/Ca(2+) exchange are amplified by a mechanism compatible with calcium-calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4-chloro-m-cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co-localization of immunofluorescent labeled Na(+)/Ca(2+) exchanger and RyRs. Low (< 50 micromol/L) or high (> 500 micromol/L) concentrations of L-glutamate (L-Glu) or L-aspartate causes a rise in which is completely blocked by the Na(+)/Ca(2+) exchange inhibitors KB-R7943 and SEA0400. The most important novel finding presented in this work is that L-Glu activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing Na(+) entry through the electrogenic Na(+)-Glu-co-transporter and not through the ionophoric L-Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoric L-Glu receptors and the electrogenic Glu transporter.     

Role of annexin A6 isoforms in catecholamine secretion by PC12 cells: Distinct influence on calcium response

Noradrenaline and adrenaline are secreted by adrenal medulla chromaffin cells via exocytosis. Exocytosis of catecholamines occurs after cell stimulation with various endogenous activators such as nicotine or after depolarization of the plasma membrane and is regulated by calcium ions. Cytosolic [Ca²⁺] increases in response to cell excitation and triggers a signal‐initiated secretion. Annexins are known to participate in the regulation of membrane dynamics and are also considered to be involved in vesicular trafficking. Some experimental evidence suggests that annexins may participate in Ca²⁺‐regulated catecholamine secretion. In this report the effect of annexin A6 (AnxA6) isoforms 1 and 2 on catecholamine secretion has been described. Overexpression of AnxA6 isoforms and AnxA6 knock‐down in PC12 cells were accompanied by almost complete inhibition or a 20% enhancement of dopamine secretion, respectively. AnxA6‐1 and AnxA6‐2 overexpression reduced Δ[Ca²⁺]_c upon depolarization by 32% and 58%, respectively, while AnxA6 knock‐down increased Δ[Ca²⁺]_c by 44%. The mechanism of AnxA6 action on Ca²⁺ signalling is not well understood. Experimental evidence suggests that two AnxA6 isoforms interact with different targets engaged in regulation of calcium homeostasis in PC12 cells. J. Cell. Biochem. 111: 168–178, 2010. © 2010 Wiley‐Liss, Inc.     

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.     

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.     

Non-additive potentiation of glutamate release by phorbol esters and metabotropic mGlu7 receptor in cerebrocortical nerve terminals

We recently showed that prolonged activation of metabotropic glutamate receptor 7 (mGlu7) potentiates glutamate release. This signalling involves phospholipase C activation via a pertussis toxin insensitive G protein and the subsequent hydrolysis of phosphatidylinositol (4,5)-bisphosphate. Release potentiation is independent of protein kinase C activation but it is dependent on the downstream release machinery, as reflected by the concomitant translocation of active zone Munc13-1 protein from the soluble to particulate fractions. Here we show that phorbol ester and mGlu7 receptor-dependent facilitation of neurotransmitter release is not additive, suggesting they share a common signalling mechanism. However, release potentiation is restricted to release sites that express N-type Ca(2+) channels, because phorbol ester and mGlu7 receptor-mediated release potentiation are absent in nerve terminals from mice lacking N-type Ca(2+) channels. In addition, phorbol esters but not mGlu7 receptors potentiate release at nerve terminals with P/Q-type Ca(2+) channels, although only under restricted conditions of Ca(2+) influx. The differential effect of phorbol esters at nerve terminals with either N- or P/Q-type Ca(2+) channels seems to be unrelated to the type Munc13 isoform expressed, and it is more likely dependent on other properties of the release machinery.     

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.