Effect of synaptosomal cytosolic [3H]GABA pool depletion on secretory ability of alpha-latrotoxin

alpha-Latrotoxin, a presynaptic neurotoxin from the venom of Latrodectus mactans tredecimguttatus, induces massive [3H]GABA release from rat brain synaptosomes as a result of interaction with either Ca(2+)-dependent (neurexin 1 alpha or Ca(2+)-independent (latrophilin) membrane receptor. The main aim of the study was to elucidate whether the binding of alpha-latrotoxin to different types of receptors led to [3H]GABA secretion from one pool or in each case the source of neurotransmitter differs: in the presence of Ca2+ exocytosis is induced, while in the absence of Ca(2+)--outflow by mobile membrane GABA transporter from cytoplasm. We examined the effect of the depletion of cytosolic [3H]GABA pool by competitive inhibitors of the GABA transporter (nipecotic acid and 2,4-diaminobutyric acid) on the alpha-latrotoxin-stimulated neurotransmitter release. We also compared the influence of these agents on neurosecretion, evoked by depolarization with that evoked by alpha-latrotoxin. Depolarization was stimulated by 4-aminopyridine in the Ca(2+)-containing saline and high KCl in Ca(2+)-free medium. In synaptosomes treated with nipecotic acid unstimulated [3H]GABA release was significantly augmented and high KCl-evoked Ca(2+)-independent [3H]GABA release was essentially inhibited. But under the same conditions neurosecretion stimulated by alpha-latrotoxin greatly raised with respect to the control response. The similar results were obtained with the synaptosomes treated with 2,4-diaminobutyric acid. Another way to determine which of GABA pool is the target of alpha-latrotoxin action lay in analysis of the toxin effects on the preliminary depolarized synaptosomes. alpha-Latrotoxin influence was diminished by the preceding depolarization by 4-aminopyridine in Ca2+ presence. But after the high KCl stimulation effect of alpha-latrotoxin didn't change. These data suggest that alpha-latrotoxin triggers neurotransmitter release from synaptic vesicles via exocytosis. We suppose that the type of membrane receptor does not determine the mechanism of GABA release evoked by the toxin.     

Does extracellular calcium determine what pool of GABA is the target for alpha-latrotoxin?

Presynaptic neurotoxin alpha-latrotoxin, from the venom of Latrodectus mactans tredecimguttatus, causes massive [(3)H]GABA release from rat brain synaptosomes, irrespective of calcium presence in the extracellular medium. Whether the binding of alpha-latrotoxin to Ca(2+)-dependent (neurexin 1 alpha) or to Ca(2+)-independent (latrophilin) receptor triggers [(3)H]GABA release by the same mechanisms or different ones, inducing either exocytotic process or outflow by mobile membrane GABA transporter, is unknown. We examined alpha-latrotoxin-evoked [(3)H]GABA release from synaptosomes which cytosolic [(3)H]GABA pool was depleted either by applying competitive inhibitors of the GABA transporter, nipecotic acid and 2,4-diaminobutyric acid, or by permeation with digitonin. We also compared the effect of the GABA transporter inhibitors on depolarisation-evoked and alpha-latrotoxin-evoked [(3)H]GABA release using as depolarising agents 4-aminopyridine and high KCl in the Ca(2+)-containing and in Ca(2+)-free medium, respectively. Incubation of synaptosomes with nipecotic acid induced the essential acceleration of unstimulated [(3)H]GABA release and deep inhibition of high KCl-evoked Ca(2+)-independent [(3)H]GABA release. In contrast, at the similar conditions the effect of alpha-latrotoxin was greatly augmented with respect to the control response. Another way to assay what GABA pool was involved in alpha-latrotoxin-induced release lays in an analysis of the effects of depolarisation and alpha-latrotoxin in consecutive order. The preliminary 4-aminopyridine-stimulated [(3)H]GABA release attenuated the toxin effect. But when depolarisation occurred in Ca(2+)-free medium, no influence on alpha-latrotoxin effect was revealed. Employing digitonin-permeated synaptosomes, we have shown that alpha-latrotoxin could stimulate [3H]GABA release in the medium with 1mM EGTA, this effect of the toxin was blocked by concanavalin A and was ATP-dependent. The latter suggests that alpha-latrotoxin-released neurotransmitter has the vesicular nature. We assume that the type of the toxin membrane receptor does not determine the mechanisms of [(3)H]GABA release evoked by alpha-latrotoxin.     

Involvement of membrane GABA transporter in alpha-latrotoxin-stimulated [3H]GABA release

Alpha-latrotoxin evokes massive [3H]GABA release from rat brain synaptosomes by stimulating exocytosis and outflow from non-vesicular pool. In the present study, GABA transporter-mediated [3H]GABA release was shown to be involved in alpha-latrotoxin-triggered release of [3H]GABA from non-vesicular pool. The following agents have been exploited as tools: (1) a protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and bafilomycin A1 for evoking depletion of synaptic vesicle [3H]GABA and enlargement of non-vesicular pool; (2) a non-substrate high-affinity GABA transport blocker NO-711 for determining participation of GABA carrier in the toxin-stimulated GABA release; (3) a competitive inhibitor of GABA reuptake nipecotic acid for heteroexchange [3H]GABA release. As shown by the experiments with nipecotic acid, FCCP and bafilomycin A1 considerably increase the content of non-vesicular [3H]GABA. The treatment of the synaptosomes with these agents modified the response to alpha-latrotoxin, particularly to its subnanomolar concentrations: the lack or substantial lowering of the toxin-evoked release during the first 2 min after the toxin addition and substantial enhancement of release up to the 5th minute were observed. Only the step of enhanced release was sensitive to GABA transporter blocker NO-711. Distinct sensitivity to NO-711 was shown to be characteristic for different steps of alpha-latrotoxin-stimulated [3H]GABA release from the control, untreated synaptosomes: lack of any effect of NO-711 during the first 2 min and powerful inhibition in 10 min after the toxin application. Taken together these data appear to indicate that the toxin non-simultaneously from vesicular and non-vesicular origins releases the neurotransmitter, the first rapid step reflects exocytosis stimulation, and the second tardy step is at least in part due to the release mediated by GABA transporters. The incomplete inhibition with NO-711 of the tardy step of the release evoked by nanomolar toxin concentrations suggests the participation not only of the GABA transporters.     

Improvement of metabolic competence of isolated nerve terminals by extracellular pyruvate

Neuronal activity is tightly coupled with brain energy metabolism. Numerous studies have proved that glucose is not a sole energy substrate for neurons; metabolic monocarboxylate intermediates derived from glucose (pyruvate and lactate) released by astrocytes are shown to be taken up and oxidized by neurons, and, moreover, could serve as neuroprotective agents. Herein, we presented the data that extracellular pyruvate (4 mM) in the presence of glucose caused the increase in synaptosomal ATP content from 3.48+/-0.30 to 4.38+/-0.23 nmol/mg of protein. This correlates with the enhanced accumulation of fluorescent dye acridine orange in the available and the recycling synaptic vesicles within the synaptosomes reflecting the improved generation of proton gradient through the synaptic vesicle membrane. We have also demonstrated the effect of extracellular pyruvate on distribution of [3H]GABA between synaptic vesicles and cytoplasm in loaded synaptosomes. To estimate [3H]GABA accumulation into the synaptic vesicles, Ca 2+-dependent 4-aminopyridine-triggered exocytotic neurotransmitter release was studied. Evaluation of cytosolic 1H]GABA pool was performed by measuring the Ca2+-independent transporter-mediated neurotransmitter release evoked by nipecotic acid or high K+. The presence of pyruvate resulted in doubled exocytotic release of [3H]GABA, and significantly attenuated Ca2+-independent release of cytosolic [3H]GABA. Together, these observations provide insight into the important role of glucose metabolic intermediate, pyruvate, in sustaining activity of vesicular inhibitory amino acid transporter and so normal inhibitory transmission. We propose to use pyruvate for keeping up synaptosomal preparations in state of metabolic stability.     

γ-Aminobutyric Acid and Glycine Modulate Each Other''s Release Through Heterocarriers Sited on the Releasing Axon Terminals of Rat CNS

The ability of gamma-aminobutyric acid (GABA) and glycine (Gly) to modulate each other's release was studied in synaptosomes from rat spinal cord, cerebellum, cerebral cortex, or hippocampus, prelabeled with [3H]GABA or [3H]Gly and exposed in superfusion to Gly or to GABA, respectively. GABA increased the spontaneous outflow of [3H]Gly (EC50, 20.8 microM) from spinal cord synaptosomes. Neither muscimol nor (-)-baclofen, up to 300 microM, mimicked the effect of GABA, which was not antagonized by either bicuculline or picrotoxin. However, the effect of GABA was counteracted by the GABA uptake inhibitors nipecotic acid and N-(4,4-diphenyl-3-butenyl)nipecotic acid. Moreover, the GABA-induced [3H]Gly release was Na+ dependent and disappeared when the medium contained 23 mM Na+. The effect of GABA was Ca2+ independent and tetrodotoxin insensitive. Conversely, Gly enhanced the outflow of [3H]GABA from rat spinal cord synaptosomes (EC50, 100.9 microM). This effect was insensitive to both strychnine and 7-chlorokynurenic acid, antagonists at Gly receptors, but it was strongly Na+ dependent. Also, the Gly-evoked [3H]GABA release was Ca2+ independent and tetrodotoxin insensitive. GABA increased the outflow of [3H]Gly (EC50, 11.1 microM) from cerebellar synaptosomes; the effect was not mimicked by either muscimol or (-)-baclofen nor was it prevented by bicuculline or picrotoxin. The GABA effect was, however, blocked by GABA uptake inhibitors and was Na+ dependent. Gly increased [3H]GABA release from cerebellar synaptosomes (EC50, 110.7 microM) in a strychnine- and 7-chlorokynurenic acid-insensitive manner. This effect was Na+ dependent. The effects of GABA on [3H]Gly release seen in spinal cord and cerebellum could be reproduced also with cerebrocortical synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

alpha-Latrotoxin affects mitochondrial potential and synaptic vesicle proton gradient of nerve terminals

Ca(2+)-independent [(3)H]GABA release induced by alpha-latrotoxin was found to consist of two sequential processes: a fast initial release realized via exocytosis and more delayed outflow through the plasma membrane GABA transporters [Linetska, M.V., Storchak, L.G., Tarasenko, A.S., Himmelreich, N.H., 2004. Involvement of membrane GABA transporters in alpha-latrotoxin-stimulated [(3)H]GABA release. Neurochem. Int. 44, 303-312]. To characterize the toxin-stimulated events attributable to the transporter-mediated [(3)H]GABA release from rat brain synaptosomes we studied the effect of alpha-latrotoxin on membrane potentials and generation of the synaptic vesicles proton gradient, using fluorescent dyes: potential-sensitive rhodamine 6G and pH-sensitive acridine orange. We revealed that alpha-latrotoxin induced a progressive dose-dependent depolarization of mitochondrial membrane potential and an irreversible run-down of the synaptic vesicle proton gradient. Both processes were insensitive to the presence of cadmium, a potent blocker of toxin-formed transmembrane pores, indicating that alpha-latrotoxin-induced disturbance of the plasma membrane permeability was not responsible to these effects. A gradual dissipation of the synaptic vesicle proton gradient closely coupled with lowering the vesicular GABA transporter activity results in a leakage of the neurotransmitter from synaptic vesicles to cytoplasm. As a consequence, there is an essential increase in GABA concentration in a soluble cytosolic pool that appears to be critical parameter for altering the mode of the plasma membrane GABA transporter operation from inward to outward. Thus, our data allow clarifying what cell processes underlain a recruitment of the plasma membrane transporter-mediated pathway in alpha-LTX-stimulated secretion.     

Ca2+-Dependent and Ca2+-Independent [3H]GABA Release from Rat Brain Synaptosomes: the Effect of Temperature

The main inhibitory neurotransmitter GABA in the mammalian brain is distributed in the nerve terminals between two pools, vesicular (synaptic vesicles) and cytosolic. GABA is released from these pools by different mechanisms; there are calcium-activated exocytotic release and calcium-independent sodium-dependent release from the cytosolic pool (resulting from the membrane GABA transporter reversal). We investigated the influence of temperature on [³H]GABA release from rat brain synaptosomes, which was induced by stimulation of both these processes. In addition, we used -latrotoxin as a stimulant of [³H]GABA release. Synaptosomes from the rat brain were used in the experiments. 4-Aminopyridine (4-AP) and high [KCl] were applied to stimulate calcium-activated and calcium-independent [³H]GABA release, respectively. 4-AP-evoked [³H]GABA release was of the same intensity at 37 and 25°C (10.1 ± 1.2 and 10.1 ± 0.8% of total [³H]GABA incorporated into the synaptosomes, respectively). The effect of 4-AP on the ⁴⁵Ca²⁺ influx into synaptosomes was also temperature-independent: 0.775 ± 0.075 and 0.725 ± 0.100 nmol/min/mg of protein at 37 and 25°C, respectively. A drop in the effect of 4-AP was observed only at 15°C. When synaptosomes were depolarized with 50 mM KCl, a temperature decrease from 37°C to 25°C resulted in a twofold drop in the [³H]GABA release, from 20.5 ± 1.4 to 10.3 ± 0.7%; at 15°C [³H]GABA release dropped to less than one-third of the norm (6.0 ± 0.5%). -Latrotoxin-stimulated [³H]GABA release was diminished from 32.5 ± 2.5 at 37°C to 17.2 ± 1.3 at 25°C and 5.9 ± 0.4% at 15°C and was not affected by the presence or absence of calcium in the medium. It seems likely that the observed effect of temperature can be interpreted as based on the temperature dependence of the -latrotoxin insertion into the membrane. It is suggested that the pattern of the temperature sensitivity of GABA release from the synaptosomes can be used as a criterion for identification of the mode of neurotransmitter release.     

Phenylarsine oxide is able to dissipate synaptic vesicle acidic pool

Phenylarsine oxide (PAO) has a number of targets in the neurons, one of them is exocytotic process. In this study, we have focused on the mechanisms of phenylarsine oxide action on Ca(2+)-dependent and Ca(2+)-independent neurotransmitter release from rat brain synaptosomes. We investigated the influence of phenylarsine oxide on: (i) l-[(14)C]glutamate and [(3)H]GABA release and uptake; (ii) plasma membrane potential using a potential-sensitive fluorescent probe rhodamine 6G; (iii) exo/endocytotic process using a pH-sensitive fluorescent probe acridine orange (AO). It has been found that phenylarsine oxide induced deacidification of synaptic vesicles. This effect was completely abolished by preliminary treatment of synaptosomes with a protonophore FCCP indicating that both reagents injured a proton electrochemical gradient. Dissipation of the proton gradient by low concentrations of phenylarsine oxide (not exceed 1 microM) did not prevent KCl-triggered exocytotic response, but essentially modified endocytotic one. At higher concentrations of phenylarsine oxide (up to 10 microM), the proton gradient dissipation was intensified and the exocytotic response was fully abolished. The reagent did not change plasma membrane potential, but depolarized mitochondria. It also caused potent inhibition of the Ca(2+)-stimulated l-[(14)C]glutamate and [(3)H]GABA release and increase the Ca(2+)-independent release of l-[(14)C]glutamate, but not of [(3)H]GABA. Disulfide-reducing reagents (dithiothreitol and beta-mercaptoethanol) completely prevented phenylarsine oxide-evoked injuries. They could also restore the initial levels of the mitochondrial potential, the exocytotic response to KCl and the release and uptake of neurotransmitters. Our data provide the evidence that phenylarsine oxide causes dissipation of synaptic vesicle acidic pool resulting in the reduction of vesicle filling and as consequence in attenuation of Ca(2+)-stimulated neurotransmitter release.     

Okadaic acid and cyclosporin A modulate [(3)H]GABA release from rat brain synaptosomes

Rat brain synaptosomes were used to investigate the effect of okadaic acid, an inhibitor of protein phosphatase 1 and 2A, and cyclosporin A, an inhibitor of protein phosphatase 2B (calcineurin), on [(3)H]GABA release. Release of [(3)H]GABA was evoked by 4-aminopyridine in the presence of calcium and by alpha-latrotoxin in the presence and absence of calcium. Pretreatment of synaptosomes with 1 microM okadaic acid reduced [(3)H]GABA release evoked by 4-aminopyridine by about 40%. The effect of alpha-latrotoxin on [(3)H]GABA release was stimulated by okadaic acid. This stimulation was equal in both media. The stimulating effect of 4-aminopyridine and alpha-latrotoxin on [(3)H]GABA release was activated when synaptosomes were pretreated with cyclosporin A. Activation of 4-aminopyridine-evoked [(3)H]GABA release was observed at 1 microM cyclosporin A, but the toxin effect was enhanced only when concentration of cyclosporin A was increased to 10 microM. The level of cyclosporin A activation depended on alpha-latrotoxin concentrations used - a higher stimulating effect of cyclosporin A was observed with lower toxin concentration. These results suggest that in calcium medium 4-aminopyridine- and alpha-latrotoxin-evoked [(3)H]GABA release was realized by different mechanisms.     

Presynaptic Mechanisms Underlying the γ-Aminobutyric Acid-Evoked Receptor-Independent Release of [3H]Norepinephrine in Rat Hippocampus

The effects of gamma-aminobutyric acid (GABA) on the spontaneous efflux of [3H]norepinephrine ([3H]NE) were studied in synaptosomes prepared from rat hippocampus and prelabelled with [3H]NE. It had been observed previously that, when synaptosomes were exposed in superfusion to GABA, the basal release of the tritiated catecholamine was enhanced, apparently with no involvement of the known GABA receptors. The mechanisms underlying this effect have now been investigated. The potency of GABA as a releaser of [3H]NE was decreased by lowering the Na+ content of the superfusion medium, and its effect disappeared at 23 mM Na+. The GABA-induced [3H]NE release was counteracted by the GABA uptake inhibitor N-(4,4-diphenyl-3-butenyl)nipecotic acid (SKF 89976A), but it was unaffected by the NE uptake blockers desmethylimipramine and nisoxetine. The GABA-induced release of [3H]NE was Ca2+-dependent and tetrodotoxin-sensitive. The data support the hypothesis that GABA provoked [3H]NE release by a novel mechanism which involves penetration into the noradrenergic nerve terminals through a GABA carrier located on the NE terminals themselves. This uptake process might be electrogenic and provoke depolarization of the nerve terminals, causing an exocytotic release of [3H]NE.     

Depolarization- and α-Latrotoxin-Induced [3H]GABA Release from the Rat Brain Synaptosomes: Effect of Phenylarsine Oxide

Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of cellular processes, including neurotransmitter release. Here we present evidence for the strong influence of an inhibitor of phosphatidylinositol 4-kinase, phenylarsine oxide, on depolarization- and -latrotoxin-evoked exocytotic release of [³H]GABA from the rat brain synaptosomes. Our data also show that subnanomolar concentrations of the toxin stimulate the process of exocytosis per se, while nanomolar toxin concentrations in addition cause neurotransmitter outflow from the cytosolic pool.     

Effect of Taurine on Neurotransmitter Release from Insect Synaptosomes

The effect of taurine on the release of [3H]acetylcholine ([3H]ACH) and [3H]gamma-aminobutyric acid ([3H]GABA) from preloaded locust synaptosomes has been studied. Veratridine (100 microM) and K+ (100 mM) both evoked [3H]ACh release and this was reduced in a concentration-dependent manner by taurine (5, 10, and 20 mM). In contrast to this, veratridine induced no observable release of [3H]GABA, and the response to K+ was slight. In the presence of taurine, however, a concentration-dependent enhancement of [3H]GABA release was observed. Since nipecotic acid (1 mM), an inhibitor of neuronal GABA uptake, also revealed [3H]GABA release induced by veratridine, it is suggested that both this effect and that of taurine are due to prevention of GABA reuptake. These results suggest that taurine may act as a neuromodulator in insects.     

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.     

Ca2+-Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes

The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 microM Pb2+ to intact synaptosomes in Ca2(+)-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2(+)-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1-50 nM (EC50 congruent to 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2-5 microM (EC50 congruent to 0.5 microM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.     

Relationship of Dihydropyridine Binding Sites with Calcium-Dependent Neurotransmitter Release in Synaptosomes

In the present work, we have studied the effect of ruthenium red (RuR), La3+ and 4-aminopyridine (4-AP) on the specific binding of (+)-[3H]PN200-110 to synaptosomes, as well as the effect of nitrendipine, nifedipine, and BAY K 8644 on gamma-[3H]aminobutyric acid [( 3H]GABA) release induced by potassium depolarization and by 4-AP in synaptosomes. Scatchard plots indicated that neither RuR nor 4-AP modifies the KD and Bmax of [3H]PN200-110 specific binding, whereas La3+ decreased the Bmax by about 25%; when the effect of the drugs on the total binding of PN200-110 was studied, a similar inhibition by La3+ was found. The calcium antagonists, nitrendipine and nifedipine, did not affect at all the potassium-stimulated release of [3H]GABA nor its release induced by 4-AP. The calcium agonist BAY K 8644 failed to affect both the spontaneous and the potassium-stimulated GABA release. Our results suggest that the binding sites of dihydropyridines in presynaptic membranes are not related to the calcium channels involved in neurotransmitter release with which RuR, La3+, and 4-AP interact.     

Amphiphilic anti-SARS-CoV-2 drug remdesivir incorporates into the lipid bilayer and nerve terminal membranes influencing excitatory and inhibitory neurotransmission

Remdesivir is a novel antiviral drug, which is active against the SARS-CoV-2 virus. Remdesivir is known to accumulate in the brain but it is not clear whether it influences the neurotransmission. Here we report diverse and pronounced effects of remdesivir on transportation and release of excitatory and inhibitory neurotransmitters in rat cortex nerve terminals (synaptosomes) in vitro. Direct incorporation of remdesivir molecules into the cellular membranes was shown by FTIR spectroscopy, planar phospholipid bilayer membranes and computational techniques. Remdesivir decreases depolarization-induced exocytotic release of L-[¹⁴C] glutamate and [³H] GABA, and also [³H] GABA uptake and extracellular level in synaptosomes in a dose-dependent manner. Fluorimetric studies confirmed remdesivir-induced impairment of exocytosis in nerve terminals and revealed a decrease in synaptic vesicle acidification. Our data suggest that remdesivir dosing during antiviral therapy should be precisely controlled to prevent possible neuromodulatory action at the presynaptic level. Further studies of neurotropic and membranotropic effects of remdesivir are necessary.     

Multiple components of synaptosomal [3H]-gamma-aminobutyric acid release resolved by a rapid superfusion system

Release of [3H]-gamma-aminobutyric acid ([3H]GABA) from rat brain synaptosomes was studied with 60-ms time resolution, using a novel rapid superfusion method. Synaptosomes were prelabeled with [3H]GABA via an associated GABA uptake system. KCl depolarization stimulated at least three distinct components of GABA release: (1) a phasic Ca-dependent component, which develops rapidly and decays with a time constant of at most 60 ms; (2) a tonic Ca-dependent component that persists after KCl depolarization is ended; (3) a Ca-independent component. The three components of GABA release are pharmacologically distinct. The phasic component was selectively blocked by 50 microM Cd2+, while the tonic component was selectively blocked by 100 microM Ni2+. The Ca-independent component was selectively blocked by nipecotic acid (IC50 = 21 microM), a known inhibitor of Na+-dependent GABA uptake. The time course and amplitude of Ca-dependent GABA release evoked by the Ca2+ ionophore A23187 were nearly identical with Ca-dependent release evoked by depolarization. This result indicates that Ca-dependent GABA release depends primarily on Ca2+ entry into the nerve terminal, and not depolarization, per se. The properties of the phasic component suggest that it is normally initiated by a voltage-sensitive Ca2+ channel that is functionally and pharmacologically distinct from those previously described. The Ca-independent component of GABA release is probably mediated by reversal of the Na-dependent, electrogenic GABA uptake system. The ability to identify multiple components of GABA release on a physiologically relevant time scale may afford a more precise definition of the mechanism of action of drugs thought to affect neurotransmission in the brain.     

alpha-Latrotoxin, acting via two Ca2+-dependent pathways, triggers exocytosis of two pools of synaptic vesicles

alpha-Latrotoxin stimulates three types of [(3)H]gamma-aminobutyric acid and [(14)C]glutamate release from synaptosomes. The Ca(2+)-independent component (i) is insensitive to SNAP-25 cleavage or depletion of vesicle contents by bafilomycin A1 and represents transmitter efflux mediated by alpha-latrotoxin pores. Two other components of release are Ca(2+)-dependent and vesicular but rely on distinct mechanisms. The fast receptor-mediated pathway (ii) involves intracellular Ca(2+) stores and acts upon sucrose-sensitive readily releasable vesicles; this mechanism is insensitive to inhibition of phosphatidylinositol 4-kinase (PI 4-kinase). The delayed pore-dependent exocytotic component (iii) is stimulated by Ca(2+) entering through alpha-latrotoxin pores; it requires PI 4-kinase and occurs mainly from depot vesicles. Lanthanum perturbs alpha-latrotoxin pores and blocks the two pore-mediated components (i, iii) but not the receptor-mediated release (ii). alpha-Latrotoxin mutant (LTX(N4C)) cannot form pores and stimulates only the Ca(2+)-dependent receptor-mediated amino acid exocytosis (ii) (detectable biochemically and electrophysiologically). These findings explain experimental data obtained by different laboratories and implicate the toxin receptors in the regulation of the readily releasable pool of synaptic vesicles. Our results also suggest that, similar to noradrenergic vesicles, amino acid-containing vesicles at some point in their cycle require PI 4-kinase.     

Effects of staurosporine on Ca2+-dependent and Ca2+-independent [14C]GABA release from rat brain synaptosomes

The effect of a protein kinase inhibitor, staurosporine, on Ca²⁺-dependent and Ca²⁺-independent release of [¹⁴C]GABA in isolated rat brain synaptosomes was studied. Calcium-dependent [¹⁴C]GABA release was stimulated by depolarization with a K⁺ channel blocker, 4-aminopyridine (4-AP), or high K⁺ concentration. It has been shown that the effect of 4-AP is Ca²⁺-dependent, while high K⁺ is able to evoke [¹⁴C]GABA release in both Ca²⁺-dependent and Ca²⁺-independent manners. In addition, Ca²⁺-independent [¹⁴C]GABA release was studied using α-latrotoxin (LTX) as a tool. Pretreatment of synaptosomes with staurosporine resulted in pronounced inhibition of 4-AP-stimulated Ca²⁺-dependent [¹⁴C]GABA release. The inhibitory effect of staurosporine on [¹⁴C]GABA release was not due to modulation of 4-AP-promoted⁴⁵Ca²⁺ influx into synaptosomes. If the process of [¹⁴C]GABA release occurred in the Ca²⁺-independent manner irrespectively of what, LTX or high K⁺, stimulated this process, it was not inhibited by staurosporine. Considering the above findings, it is reasonable to assume that the absence of Ca²⁺ in the extracellular medium created conditions for activation of the process of neurotransmitter release without Ca²⁺-dependent dephosphorylation of neuronal phosphoproteins; as a consequence, regulation of exocytotic process was modulated in such a manner that inhibition of protein kinases did not disturb exocytosis.     

Influence of Integrin-blocking Peptide on Gadolinium- and Hypertonic Shrinking-induced Neurotransmitter Release in Rat Brain Synaptosomes

Polyvalent cations and hypertonic shrinking of presynaptic endings lead to calcium-independent exocytosis in various synapses. In the present study we have investigated the contribution of integrins to this phenomenon. It was found that hypertonic shrinking, polyvalent cations ruthenium red and gadolinium results in dose-dependent calcium-independent neurotransmitter release in rat brain synaptosomes. The exocytotic mechanism of neurotransmitter release induced by 300 microM gadolinium was additionally verified by the fluorescent dye FM2-10. We found that 200 microM of RGDS peptide, an inhibitor of integrins, decreased polyvalent gadolinium-induced [3H]D: -aspartate release by 26%. This compound had no effect upon hypertonicity-induced release. The peptide RGES, a negative control for RGDS; genistein, an inhibitor of tyrosine kinases; and citrate, an inhibitor of lanthanides-induced aggregation were ineffective in both cases. Therefore, we have shown that integrins did not influence hypertonicity-evoked [3H]D: -aspartate release, but partially mediated that evoked by gadolinium ions.