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A novel tool for the investigation of glutamate release from rat cerebrocortical synaptosomes: the toxin Tx3-3 from the venom of the spider Phoneutria nigriventer.

ABSTRACT: The present experiments investigated the effect of some of the toxic components present in the venom of the spider Phoneutria nigriventer on the release of neurotransmitter. The toxic fraction, Phoneutria nigriventer toxin-3 (PhTx3), abolished Ca2+-dependent glutamate release, but did not alter Ca2+-independent secretion of glutamate when rat brain cortical synaptosomes were depolarized with 33 mM KCl. This effect was most likely due to interference with the entry of calcium through voltage-gated calcium channels, because PhTx3 reduced by 50% the increase in intrasynaptosomal free calcium induced by membrane depolarization, and did not affect the release of glutamate evoked by a calcium ionophore (ionomycin). A polypeptide (Tx3-3) present in the PhTx3 fraction reproduced the effects of the PhTx3 fraction on transmitter release and intrasynaptosomal free calcium in the low nanomolar range. We compared the alterations produced by the Tx3-3 with the actions of toxins known to block calcium channels coupled to exocytosis: the results indicated that the Tx3-3 inhibition of glutamate release and intrasynaptosomal calcium resemble that observed with omega-conotoxin MVIIC. We suggest that the Tx3-3 is a calcium-channel antagonist that blocked glutamate exocytosis.

SUBMITTER: Prado MA

PROVIDER: S-EPMC1217017 | biostudies-other | 1996 Feb

REPOSITORIES: biostudies-other

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Leucine-rich repeat kinase 2 (LRRK2) inhibitors differentially modulate glutamate release and Serine935 LRRK2 phosphorylation in striatal and cerebrocortical synaptosomes.

Project description:Mutations in leucine-rich repeat kinase 2 (LRRK2) gene have been pathogenically linked to Parkinson's disease, and pharmacological inhibition of LRRK2 is being pursued to tackle nigro-striatal dopaminergic neurodegeneration. However, LRRK2 kinase inhibitors may have manifold actions, affecting not only pathological mechanisms in dopaminergic neurons but also physiological functions in nondopaminergic neurons. Therefore, we investigated whether LRRK2 kinase inhibitors differentially modulate dopamine and glutamate release from the mouse striatum and cerebral cortex. Spontaneous and KCl-evoked [3H]-dopamine and glutamate release from superfused synaptosomes obtained from wild-type and LRRK2 knock-out, kinase-dead or G2019S knock-in mice was measured. Two structurally unrelated inhibitors, LRRK2-IN-1 and GSK2578215A, were tested. LRRK2, phosphoSerine1292 and phosphoSerine935 LRRK2 levels were measured in all genotypes, and target engagement was evaluated by monitoring phosphoSerine935 LRRK2. LRRK2-IN-1 inhibited striatal glutamate but not dopamine release; GSK2578215A inhibited striatal dopamine and cortical glutamate but enhanced striatal glutamate release. LRRK2-IN-1 reduced striatal and cortical phosphoSerine935 levels whereas GSK2578215A inhibited only the former. Neither LRRK2 inhibitor affected neurotransmitter release in LRRK2 knock-out and kinase-dead mice; however, they facilitated dopamine without affecting striatal glutamate in G2019S knock-in mice. GSK2578215A inhibited cortical glutamate release in G2019S knock-in mice. We conclude that LRRK2-IN-1 and GSK2578215A modulate exocytosis by blocking LRRK2 kinase activity, although their effects vary depending on the nerve terminal examined. The G2019S mutation unravels a dopamine-promoting action of LRRK2 inhibitors while blunting their effects on glutamate release, which highlights their positive potential for the treatment of PD, especially of LRRK2 mutation carriers.

| S-EPMC6536420 | biostudies-literature

Rosmarinic Acid, a Bioactive Phenolic Compound, Inhibits Glutamate Release from Rat Cerebrocortical Synaptosomes through GABA<sub>A</sub> Receptor Activation.

Project description:Rosmarinic acid, a major component of rosemary, is a polyphenolic compound with potential neuroprotective effects. Asreducing the synaptic release of glutamate is crucial to achieving neuroprotectant's pharmacotherapeutic effects, the effect of rosmarinic acid on glutamate release was investigated in rat cerebrocortical nerve terminals (synaptosomes). Rosmarinic acid depressed the 4-aminopyridine (4-AP)-induced glutamate release in a concentration-dependent manner. The removal of extracellular calcium and the blockade of vesicular transporters prevented the inhibition of glutamate release by rosmarinic acid. Rosmarinic acid reduced 4-AP-induced intrasynaptosomal Ca2+ elevation. The inhibition of N-, P/Q-type Ca2+ channels and the calcium/calmodulin-dependent kinase II (CaMKII) prevented rosmarinic acid from having effects on glutamate release. Rosmarinic acid also reduced the 4-AP-induced activation of CaMKII and the subsequent phosphorylation of synapsin I, the main presynaptic target of CaMKII. In addition, immunocytochemistry confirmed the presence of GABAA receptors. GABAA receptor agonist and antagonist blocked the inhibitory effect of rosmarinic acid on 4-AP-evoked glutamate release. Docking data also revealed that rosmarinic acid formed a hydrogen bond with the amino acid residues of GABAA receptor. These results suggested that rosmarinic acid activates GABAA receptors in cerebrocortical synaptosomes to decrease Ca2+ influx and CaMKII/synapsin I pathway to inhibit the evoked glutamate release.

| S-EPMC8301814 | biostudies-literature

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| S-EPMC4394492 | biostudies-literature

Effects of adenosine A1 and A2A receptor activation on the evoked release of glutamate from rat cerebrocortical synaptosomes.

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| S-EPMC1573357 | biostudies-other

Modulatory features of the novel spider toxin ?-TRTX-Df1a isolated from the venom of the spider Davus fasciatus.

Project description:BACKGROUND AND PURPOSE:Naturally occurring dysfunction of voltage-gated sodium (NaV ) channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel NaV modulators, we investigated spider venoms for new inhibitors of NaV channels. EXPERIMENTAL APPROACH:We used high-throughput screens to identify a NaV modulator in venom of the spider Davus fasciatus. Further characterization of this venom peptide was undertaken using fluorescent and electrophysiological assays, molecular modelling and a rodent pain model. KEY RESULTS:We identified a potent NaV inhibitor named ?-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by NaV 1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited voltage-gated calcium (CaV 3) currents but had no activity against the voltage-gated potassium (KV 2) channel. The modelled structure of Df1a, which contains an inhibitor cystine knot motif, is reminiscent of the NaV channel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all NaV subtypes tested (hNaV 1.1-1.7). Df1a also slowed fast inactivation of NaV 1.1, NaV 1.3 and NaV 1.5 and modified the voltage-dependence of activation and inactivation of most of the NaV subtypes. Df1a preferentially binds to the domain II voltage-sensor and has additional interactions with the voltage sensors domains III and IV, which probably explains its modulatory features. Df1a was analgesic in vivo, reversing the spontaneous pain behaviours induced by the NaV activator OD1. CONCLUSION AND IMPLICATIONS:?-TRTX-Df1a shows potential as a new molecule for the development of drugs to treat pain disorders mediated by voltage-gated ion channels.

| S-EPMC5513869 | biostudies-literature

Novel class of spider toxin: active principle from the yellow sac spider Cheiracanthium punctorium venom is a unique two-domain polypeptide.

Project description:Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a-c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.

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Purification and Characterization of a Novel Insecticidal Toxin, μ-sparatoxin-Hv2, from the Venom of the Spider Heteropoda venatoria.

Project description:The venom of the spider Heteropoda venatoria produced lethal effect to cockroaches as reported in our previous study, and could be a resource for naturally-occurring insecticides. The present study characterized a novel cockroach voltage-gated sodium channels (NaVs) antagonist, μ-sparatoxin-Hv2 (μ-SPRTX-Hv2 for short), from this venom. μ-SPRTX-Hv2 is composed of 37 amino acids and contains six conserved cysteines. We synthesized the toxin by using the chemical synthesis method. The toxin was lethal to cockroaches when intraperitoneally injected, with a LD50 value of 2.8 nmol/g of body weight. Electrophysiological data showed that the toxin potently blocked NaVs in cockroach dorsal unpaired median (DUM) neurons, with an IC50 of 833.7 ± 132.2 nM, but it hardly affected the DUM voltage-gated potassium channels (KVs) and the DUM high-voltage-activated calcium channels (HVA CaVs). The toxin also did not affect NaVs, HVA CaVs, and Kvs in rat dorsal root ganglion (DRG) neurons, as well as NaV subtypes NaV1.3⁻1.5, NaV1.7, and NaV1.8. No envenomation symptoms were observed when μ-SPRTX-Hv2 was intraperitoneally injected into mouse at the dose of 7.0 μg/g. In summary, μ-SPRTX-Hv2 is a novel insecticidal toxin from H. venatoria venom. It might exhibit its effect by blocking the insect NaVs and is a candidate for developing bioinsecticide.

| S-EPMC6024679 | biostudies-literature

A novel neurotoxin from venom of the spider, Brachypelma albopilosum.

Project description:Spiders have evolved highly selective toxins for insects. There are many insecticidal neurotoxins in spider venoms. Although a large amount of work has been done to focus on neurotoxicity of spider components, little information, which is related with effects of spider toxins on tumor cell proliferation and cytotoxicity, is available for Brachypelma albopilosum venom. In this work, a novel spider neurotoxin (brachyin) was identified and characterized from venoms of the spider, Brachypelma albopilosum. Brachyin is composed of 41 amino acid residues with the sequence of CLGENVPCDKDRPNCCSRYECLEPTGYGWWYASYYCYKKRS. There are six cysteines in this sequence, which form three disulfided bridges. The serine residue at the C-terminus is amidated. Brachyin showed strong lethal effects on American cockroaches (Periplaneta americana) and Tenebrio molitor (common mealbeetle). This neurotoxin also showed significant analgesic effects in mice models including abdominal writhing induced by acetic acid and formalin-induced paw licking tests. It was interesting that brachyin exerted marked inhibition on tumor cell proliferation.

| S-EPMC4203764 | biostudies-literature

Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies Against Loxosceles Spider Venom.

Project description:Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen's formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.

| S-EPMC6409891 | biostudies-literature

A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery.

Project description:Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis based on synthetic gene fragments is one of the most promising emerging technologies, theoretically allowing the rapid, laboratory-scale production of specific venom components, but this approach has yet to be applied in venom biodiscovery. Here, we tested the ability of three commercially available cell-free protein expression systems to produce venom components from small arthropods, using U2-sicaritoxin-Sdo1a from the six-eyed sand spider Hexophtalma dolichocephala as a case study. We found that only one of the systems was able to produce an active product in low amounts, as demonstrated by SDS-PAGE, mass spectrometry, and bioactivity screening on murine neuroblasts. We discuss our findings in relation to the promises and limitations of cell-free synthesis for venom biodiscovery programs in smaller invertebrates.

| S-EPMC8402385 | biostudies-literature

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