Selective block of Ca2+-dependent K+ current in crayfish neuromuscular system and chromaffin cells by sea anemone Bunodosoma cangicum venom

The effects of the nematocyst venom of the sea anemone Bunodosoma cangicum on depolarization-activated currents were studied in opener crayfish muscle fibers and in cultured bovine chromaffin cells. The venom selectively and reversibly blocked the Ca²⁺ -dependent K⁺ current (I_K(Ca)) present in crayfish muscle in a dose-dependent manner without affecting voltage-gated Ca²⁺ or K⁺ currents. Furthermore, the venom also reduced I_K(Ca) in chromaffin cells, without modifying voltage-gated Na⁺, Ca²⁺, or K⁺ currents. Synaptic transmission in crayfish muscle was also affected by the venom. Repetitive excitatory and inhibitory postsynaptic currents (each associated with a presynaptic action potential) were evoked by each nerve stimulus, suggesting that presynaptic I_K(Ca) may control the electrical activity of excitatory and inhibitory presynaptic fibers. We conclude that B. cangicum venom includes a toxin that selectively and reversibly blocks Ca²⁺ -dependent K⁺ currents in crayfish muscle and in bovine chromaffin cells, and modifies excitatory and inhibitory synaptic transmission, probably abolishing a similar conductance at the presynaptic fibers. © 1995 Wiley-Liss, Inc.     

Synaptosomal glutamate release and uptake in mice lacking the cellular prion protein

Glutamate plays a central role in the fast excitatory synaptic transmission and is a key neurotransmitter involved in several neurophysiological processes. Glutamate levels on the synaptic cleft are related to neural excitability, neuroplasticity, and neuronal damage associated with excitotoxicity. Mice lacking the cellular prion protein (PrP(c)) gene (Prnp) present a decreased astrocytic glutamate uptake in cultures, higher neuronal excitability in vitro and sensitivity to pro-convulsant drugs in vivo, and age-dependent memory impairment. Here, we investigate if PrP(c) might be involved in neuronal uptake and release of glutamate. For this purpose, we compared synaptosomal preparations from the cerebral cortex, entorhinal cortex, hippocampus, cerebellum, and olfactory bulb of 3- or 9-month-old PrP(c) null mice and with respective wild-type controls. Although we observed differences in synaptosomal glutamate release and uptake regarding the age of mice and the brain structure studied, these differences were similar for PrP(c) null mice and their respective wild-type controls. Therefore, despite a possible correlation between neuronal glutamate transporters, excitability, and neuronal damage, our results suggest that PrP(c) expression is not critical for neuronal glutamate transport.     

Single channel studies of non-competitive antagonism of a quisqualate-sensitive glutamate receptor by argiotoxin636--a fraction isolated from orb-web spider venom

The effects of purified spider toxin (argiotoxin636) on single glutamate-activated channels in voltage-clamped locust muscle fibres have been examined using a megaohm seal, patch-clamp technique. Four experimental protocols were employed in which the composition of the patch pipette and bathing solutions were varied. Three types of channel behaviour were broadly defined when argiotoxin636 was present either in the patch pipette or in the muscle bath; the type of channel behaviour being dependent upon the concentration of argiotoxin636 and/or the duration of its application. Type I behaviour was characterized by reductions in channel open probability (Po) and channel event frequency (f), by an increase in mean channel closed time (mc) and either no change in mean channel open time (mo) or, infrequently, an increase in this parameter; Type II behaviour was characterized by apparent absence of channel openings. For example, with 10(-12) M argiotoxin636 in the patch pipette Type I behaviour changed to Type II behaviour after approximately 60 s and from Type II behaviour to Type III behaviour after approximately 120 s. The results of this study are consistent with the idea that argiotoxin636 blocks the cation-selective channel gated by excitatory glutamate receptors in insect muscle at the level of the open channel although there remains the possibility that it is also either a closed channel blocker and/or a competitive antagonist. The increase in mo seen in a few recordings during the initial stage of argiotoxin636 antagonism raises the possibility that the toxin interacts allosterically with the glutamate binding sites on the excitatory glutamate receptor.     

PP2 inhibits glutamate release from nerve endings by affecting vesicle mobilization

Src kinase is widely expressed in the brain and its inhibition with PP2 has previously been shown to depress depolarization-evoked glutamate release from rat cerebrocortical synaptosomes by reducing voltage-dependent Ca2+ entry. In this study, we further showed that the inhibitory effect of PP2 on 4-aminopyridine-evoked glutamate release results from a reduction of vesicular exocytosis and not from an inhibition of non-vesicular release. In addition, PP2 significantly inhibited ionomycin-induced or hypertonic sucrose-induced glutamate release. Also, disruption of cytoskeleton organization with cytochalasin D occluded the inhibitory action of PP2 on 4-aminopyridine and ionomycin-evoked glutamate release. These results suggest that PP2-mediated inhibition of glutamate release involves the modulation of some exocytotic steps, possibly through a regulation of actin cytoskeleton dynamics.     

海葵毒素对神经胶质瘤细胞的凋亡诱导作用

目的 研究从海葵组织中提取的海葵毒素(Phyllodiscus semonii toxin,PsTX)对人神经胶质瘤细胞(U251)凋亡的诱导作用及其可能机制.方法 MTT法检测PsTX对肿瘤细胞的增殖抑制率;原位末端脱氧核糖苷肽转移酶分析法(TUNEL)及DNA Ladder法检测PsTX对肿瘤细胞的凋亡诱导作用;免疫组织化学染色显示Fas蛋白在U251细胞中的表达.结果 PsTX对人神经胶质瘤细胞具有明显的生长抑制及促凋亡作用,PsTX诱导Fas在人神经胶质瘤细胞膜上表达增高.结论 PsTX可能通过Fas途径诱导人神经胶质瘤细胞凋亡.     

Vitamin B2 inhibits glutamate release from rat cerebrocortical nerve terminals

We examined the effect of riboflavin, vitamin B2, on the release of endogenous glutamate from nerve terminals purified from rat cerebral cortex. The release of glutamate evoked by 4-aminopyridine was inhibited by riboflavin. Further experiments indicated that riboflavin-mediated inhibition of glutamate release (i) results from a reduction of vesicular exocytosis, not from an inhibition of nonvesicular release; (ii) is associated with a decrease in presynaptic N-type and P/Q-type voltage-dependent Ca channel activity. These findings are the first to suggest that, in rat cerebrocortical nerve terminals, riboflavin suppresses voltage-dependent Ca channel activity and in so doing inhibits evoked glutamate release. This finding may explain the neuroprotective effects of vitamin B2 against neurotoxicity.     

Anticonvulsant action of an arylamine-containing fraction from Agelenopsis spider venom

A low molecular weight fraction (AG2) containing arylamine compounds has been isolated from venom of the spider Agelenopsis aperta. When administered intravenously or intracerebroventricularly, AG2 produces dose-dependent suppression of behavioral convulsions induced in rats by kainic acid, picrotoxin, or bicuculline. The low molecular weight compounds in spider venoms may provide novel tools for anticonvulsant research and therapy.     

Enhancement by GABA of glutamate depolarization-induced release from cerebellar nerve endings

Cerebellar synaptosomes were superfused in the presence of D-[3H]aspartate (to label the glutamate 'reuptake pool') and [14C]glutamine (to label the 'new synthesis pool'). The depolarization-induced release of D-[3H]aspartate and of newly synthesized [14C]glutamate were potentiated by low concentrations of GABA (2--20 microM) or muscimol. The effect was probably mediated by the interaction of GABA with presynaptic GABA receptors localized in 'glutamergic' nerve endings, since it was antagonized by the GABA antagonists picrotoxin and bicuculline.     

Deficient NMDA-mediated glutamate release from synaptosomes of schizophrenics

Previous studies from our laboratory indicated that the veratridine-induced release of glutamate and GABA from synaptosomes derived from brains of schizophrenics was decreased. In the present study, synaptosomes were prepared from frozen brain samples from schizophrenics and from controls. Stimulation by 10 μmol/L 2-amino-3-hydroxy-5-methoxylisoxazole-4-propionic acid (AMPA) produced equal glutamate release from both groups. Release induced by either 10 μmol/L kainic acid (KA) or n-methyl-d-asparate (NMDA) was reduced significantly in the preparations derived from schizophrenics. Similarly, the amount of GABA released by 50 μmol/L glutamate was also reduced in the schizophrenic-derived synaptosomes. However, in membranes derived from the crude synaptosomal pellet, no differences between the controls and schizophrenics were observed in measures of total glutamate binding or its displacement by NMDA. The data demonstrate a deficiency in NMDA (and possibly KA) receptor functioning in schizophrenics and support the “second-generation” theories of schizophrenia as a glutamatergic deficiency disorder.     

Dual regulation of Ca2+‐dependent glutamate release from astrocytes: Vesicular glutamate transporters and cytosolic glutamate levels

Vesicular glutamate transporters (VGLUTs) are responsible for vesicular glutamate storage and exocytotic glutamate release in neurons and astrocytes. Here, we selectively and efficiently overexpressed individual VGLUT proteins (VGLUT1, 2, or 3) in solitary astrocytes and studied their effects on mechanical stimulation‐induced Ca²⁺‐dependent glutamate release. Neither VGLUT1 nor VGLUT2 overexpression changed the amount of glutamate release, whereas overexpression of VGLUT3 significantly enhanced Ca²⁺‐dependent glutamate release from astrocytes. None of the VGLUT overexpression affected mechanically induced intracellular Ca²⁺ increase. Inhibition of glutamine synthetase activity by L ‐methionine sulfoximine in astrocytes, which leads to increased cytosolic glutamate concentration, greatly increased their mechanically induced Ca²⁺‐dependent glutamate release, without affecting intracellular Ca²⁺ dynamics. Taken together, these data indicate that both VGLUT3 and the cytosolic concentration of glutamate are key limiting factors in regulating the Ca²⁺‐dependent release of glutamate from astrocytes. © 2009 Wiley‐Liss, Inc.     

Synaptosomal localization and release of glucagon-like materials in the rat brain

The subcellular localization of glucagon-like materials in the thalamus-hypothalamus and brain stem of the rat was investigated. Both glucagon immunoreactivity (GI) determined by C-terminal specific antibody and glucagon-like immunoreactivity (GLI) determined by non-specific antibody were enriched in the microsomal and synaptosomal fractions relative to the nuclear, myelin and mitochondrial fractions. Furthermore, the synaptosomal fraction of both the thalamus-hypothalamus and brain stem incubated in Krebs-Ringer bicarbonate buffer with 55 mM K+ at 37 degrees C released GI and GLI in the presence of Ca++. These findings suggested that glucagon-like substances detected in the brain have a role in the synaptic function.     

Glutaminase inhibition and the release of neurotransmitter glutamate from synaptosomes

Cerebrocortical synaptosomes were incubated with glutamine together with 6-diazo-5-oxo-L-norleucine (DON) and NH4Cl (which are known to inhibit phosphate-stimulated glutaminase) in order to assess the effect of such inhibition on the pool sizes and extent of evoked release of endogenous amino acids, particularly glutamate. DON (5 mM) inhibited glutaminase by 73-89% and NH4Cl (1-4 mM) inhibited the enzyme by 45-53% under the conditions employed. NH4Cl (4 mM) incubated with synaptosomes for 30 min reduced pool sizes of aspartate and glutamate by 28% and inhibited release of glutamate by 55% compared to control release. DON caused a decrease in both the pool size of glutamate (22%) and the extent of veratrine-evoked release of amino acids (21%).     

Vinpocetine inhibits glutamate release induced by the convulsive agent 4-aminopyridine more potently than several antiepileptic drugs

4-Aminopyridine (4-AP) is a convulsing agent that in vivo preferentially releases Glu, the most important excitatory amino acid neurotransmitter in the brain. Here the ionic dependence of 4-AP-induced Glu release and the effects of several of the most common antiepileptic drugs (AEDs) and of the new potential AED, vinpocetine on 4-AP-induced Glu release were characterized in hippocampus isolated nerve endings pre-loaded with labelled Glu ([³H]Glu). 4-AP-induced [³H]Glu release was composed by a tetrodotoxin (TTX) sensitive and external Ca²⁺ dependent fraction and a TTX insensitive fraction that was sensitive to the excitatory amino acid transporter inhibitor, TBOA. The AEDs: carbamazepine, phenytoin, lamotrigine and oxcarbazepine at the highest dose tested only reduced [³H]Glu release to 4-AP between 50–60%, and topiramate was ineffective. Vinpocetine at a much lower concentration than the above AEDs, abolished [³H]Glu release to 4-AP. We conclude that the decrease in [³H]Glu release linked to the direct blockade of presynaptic Na⁺ channels, may importantly contribute to the anticonvulsant actions of all the drugs tested here (except topiramate); and that the significantly greater vinpocetine effect in magnitude and potency on [³H]Glu release when excitability is exacerbated like during seizures, may involve the increase additionally exerted by vinpocetine in some K⁺ channels permeability.     

No enhancement by nitric oxide of glutamate release from P2 and P3 synaptosomes of rat hippocampus

Effects of nitric oxide on glutamate (Glu) release in long-term potentiation (LTP) were investigated by superfusion of conventional (P2) and large (P3) synaptosomes prepared from the rat hippocampus. Basal releasing rates of endogenous Glu from P2 and P3 fractions were 103.6 and 85.2 pmol/min/mg protein, respectively. Exposure to a depolarizing concentration of KCl (30 mM) evoked 3.58- and 4.52-fold increases in releasing rates of Glu from P2 and P3 fractions, respectively. Although the perfusion with sodium nitroprusside (NP, 10⁻³ M), a nitric oxide-releasing agent, failed to augment the K⁺-evoked releases of Glu from P2 and P3 synaptosomes, NP enhanced that from slices of the hippocampus by 39% without changing basal release. Similarly, 8-bromoguanosine3′ : 5′-cyclic monophosphate (10⁻⁴ M) increased the K⁺-evoked release of Glu from slices by 30%, but not from either synaptosomes. When synaptosomes were prepared from the hippocampus which was pretreated with two trains of electrical field stimulation (100 Hz, 0.1 ms, for 2 s), K⁺-evoked releases of Glu from P2 and P3 synaptosomes were increased by 15% and 23%, respectively. Although nitric oxide is postulated to function as a retrograde messenger to maintain LTP, present results suggest that nitric oxide may not directly act upon nerve terminals to enhance glutamate release, but that interventions of glias and short neurons may be involved in the presynaptic mechanism of LTP.     

Miniature release events of glutamate from hippocampal neurons are influenced by the dystonia-associated protein torsinA

TorsinA is an evolutionarily conserved AAA+ ATPase, and human patients with an in‐frame deletion of a single glutamate (ΔE) codon from the encoding gene suffer from autosomal‐dominant, early‐onset generalized DYT1 dystonia. Although only 30–40% of carriers of the mutation show overt motor symptoms, most experience enhanced excitability of the central nervous system. The cellular mechanism responsible for this change in excitability is not well understood. Here we show the effects of the ΔE‐torsinA mutation on miniature neurotransmitter release from neurons. Neurotransmitter release was characterized in cultured hippocampal neurons obtained from wild‐type, heterozygous, and homozygous ΔE‐torsinA knock‐in mice using two approaches. In the first approach, patch‐clamp electrophysiology was used to record glutamate‐mediated miniature excitatory postsynaptic currents (mEPSCs) in the presence of the Na⁺ channel blocker tetrodotoxin (TTX) and absence of GABA_A receptor antagonists. The intervals between mEPSC events were significantly shorter in neurons obtained from the mutant mice than in those obtained from wild‐type mice. In the second approach, the miniature exocytosis of synaptic vesicles was detected by imaging the unstimulated release of FM dye from the nerve terminals in the presence of TTX. Cumulative FM dye release was higher in neurons obtained from the mutant mice than in those obtained from wild‐type mice. The number of glutamatergic nerve terminals was also assessed, and we found that this number was unchanged in heterozygous relative to wild‐type neurons, but slightly increased in homozygous neurons. Notably, in both heterozygous and homozygous neurons, the unitary synaptic charge during each mEPSC event was unchanged. Overall, our results suggest more frequent miniature glutamate release in neurons with ΔE‐torsinA mutations. This change may be one of the underlying mechanisms by which the excitability of the central nervous system is enhanced in the context of DYT1 dystonia. Moreover, qualitative differences between heterozygous and homozygous neurons with respect to certain synaptic properties indicate that the abnormalities observed in homozygotes may reflect more than a simple gene dosage effect. Synapse 66:807–822, 2012. © 2012 Wiley Periodicals, Inc.     

Unexpected inhibitory regulation of glutamate release from rat cerebrocortical nerve terminals by presynaptic 5-hydroxytryptamine-2A receptors

Presynaptic 5-HT(2A) receptor modulation of glutamate release from rat cerebrocortical nerve terminals (synaptosomes) was investigated by using the 5-HT(2A/2C) receptor agonist (+/-)-1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI). DOI potently inhibited 4-aminopyridine (4AP)-evoked glutamate release. Involvement of presynaptic 5-HT(2A) receptors in this modulation of 4AP-evoked release was confirmed by blockade of the DOI-mediated inhibition by the 5-HT(2A) receptor antagonist ketanserin but not by the 5-HT(2C) receptor antagonist RS102221. Inhibition of glutamate release by DOI was associated with a reduction of 4AP-evoked depolarization and downstream elevation of cytoplasmic free calcium concentration ([Ca(2+)](C)) mediated via P/Q- and N-type voltage-dependent Ca(2+) channels (VDCCs). In contrast to the DOI effect on 4AP-evoked release, the agonist had no effect on high external [K(+)] (30 mM)-induced (KCl) stimulation of VDCCs or glutamate release. Likewise, release mediated by direct Ca(2+) entry with Ca(2+) ionophore (ionomycin) or by hypertonic sucrose was unaffected by DOI. Mechanistically, DOI modulation of 4AP-evoked glutamate release appeared to involve a phospholipase C/protein kinase C signaling cascade, insofar as pretreatment of synaptosomes with the phospholipase C inhibitor U73122 or protein kinase C inhibitors Ro320432 or GF109203X all effectively occluded the inhibitory effect of the agonist. Together, these results suggest that presynaptic 5-HT(2A) receptors present on glutamatergic terminals effect an unexpected depression of glutamate release by negatively modulating nerve terminal excitability and downstream VDCC activation through a signaling cascade involving phospholipase C/protein kinase C. These observations invoke presynaptic inhibitory 5-HT(2A) receptor function as a potential target for drugs to mitigate the effects of excessive glutamatergic transmission.     

Effect of toxin from the sea anemone Homostichantus duerdemi on the sodium inward current in mammalian neurons

The effect of toxin from sea anemone Homostichantus duerdemi (HTX-1) on the inward sodium current was studied on the isolated neurons of spinal ganglia of a rat. The experiments were carried out under conditions of voltage clamp and intracellular perfusion at 20 degree C. HTX-1, when added to the extracellular solution, slowed down the inactivation process of sodium channels with no action on activation process. The dependence of the fraction of modified channels upon the concentration of HTX-1 could be described by Langmuir isotherm with the dissociation constant 1.1 +/- 0.1.10(-7) M (at holding potential --100 mV). HTX-1 caused an increase in the peak of early inward current up to 80%. The binding of HTX-1 with sodium channel was strongly potential-dependent. After a short time of application (up to 5 min) the effect of HTX-1 was completely reversible. However, if the action of the toxin exceeded 30 min, the following washing for 90 min did not lead to pronounced removal of the effect.     

Evoked release of endogenous adenosine from rat cortical slices by K and glutamate

Release of endogenous adenosine from rat cortical slices was determined in response to depolarization by 30 mM K+ and by exposure to glutamate. K+ and glutamate both released adenosine. Glutamate-evoked release was decreased by approximately 50% in the presence of the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonovaleric acid (APV). The adenosine released by glutamate could modulate neurotransmission, and may have a protective effect in pathologic conditions of excess excitation involving glutamate.