Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.69 (botulinum neurotoxin)
 1,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The exocytotic release of L-glutamate from guinea-pig cerebral cortical synaptosomes can be extensively inhibited by preincubation with botulinum neurotoxin type A at 37 degrees C for 1-2 h. The toxin has no effect on synaptosomal respiratory control, respiratory capacity, ATP synthesis, plasma-membrane 86Rb+ permeability or plasma-membrane potential, does not inhibit the entry of 45Ca2+ into the synaptosome upon depolarization and does not alter the ability of intrasynaptosomal mitochondria to sequester Ca2+. The blockade of Ca2+-dependent glutamate release may be totally reversed by the Ca2+/2 H+-exchange ionophore ionomycin, but not by increasing extracellular Ca2+ concentration. It is suggested (a) that exocytosis is triggered by the penetration of Ca2+ into an intracellular hydrophobic milieu; (b) that this stage is blocked by the toxin and (c) that ionomycin is able to bypass this block and deliver Ca2+ to the exocytotic apparatus.
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PMID:Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes. 243 34

We investigated the effect of poisoning rat brain synaptosomes with botulinum neurotoxin A on the NSF-mediated disassembly of a complex consisting of syntaxin, SNAP-25 and synaptobrevin (fusion complex). Botulinum neurotoxin A specifically removes 9 amino acids from the C-terminus of SNAP-25 and efficiently blocks KCl-evoked glutamate release from synaptosomes. We report that truncated SNAP-25 is incorporated into the fusion complex of poisoned synaptosomes. The presence of truncated SNAP-25 does not interfere with the NSF-induced disassembly of the fusion complex. Also, the release of truncated SNAP-25 from the fusion complex is similar to that of the native SNAP-25. Since neither the formation of the complex nor its disassembly seems to be affected by the SNAP-25 fragment, this fragment is likely to block exocytosis by disrupting events between disassembly of the synaptosomal fusion complex and membrane fusion itself.
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PMID:Poisoning by botulinum neurotoxin A does not inhibit formation or disassembly of the synaptosomal fusion complex. 762 35

Rat brain synaptosomes were used to study the effect of several clostridial neurotoxins on the neurotransmitter release. In this system the blockade of transmitter release correlated with the proteolytic activity of the toxins. Blockade of glutamate release was linked to selective proteolysis of one of the following synaptic proteins: synaptobrevin (BoNT/D, BoNT/F); SNAP-25 (BoNT/A, BoNT/E), or HPC-1/syntaxin (BoNT/C1). All the toxins used had an inhibitory effect on synaptosomes with the exception of BoNT/F. BoNT/F cleaved synaptobrevin in permeabilized synaptosomes but failed to produce the same effect on intact synaptosomes.
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PMID:Inhibition of neurotransmitter release by clostridial neurotoxins correlates with specific proteolysis of synaptosomal proteins. 787 84

Neurotransmitter release is potently blocked by a group of structurally related toxin proteins produced by Clostridium botulinum. Botulinum neurotoxin type B (BoNT/B) and tetanus toxin (TeTx) are zinc-dependent proteases that specifically cleave synaptobrevin (VAMP), a membrane protein of synaptic vesicles. Here we report that inhibition of transmitter release from synaptosomes caused by botulinum neurotoxin A (BoNT/A) is associated with the selective proteolysis of the synaptic protein SNAP-25. Furthermore, isolated or recombinant L chain of BoNT/A cleaves SNAP-25 in vitro. Cleavage occurred near the carboxyterminus and was sensitive to divalent cation chelators. In addition, a glutamate residue in the BoNT/A L chain, presumably required to stabilize a water molecule in the zinc-containing catalytic centre, was required for proteolytic activity. These findings demonstrate that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25. Thus, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.
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PMID:Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. 810 14

1. Presynaptic inhibition of synaptic transmission in the hippocampus was investigated by comparing the effects of several agonists on miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs). 2. The Ca2+ ionophore ionomycin increased the frequency of mEPSCs and mIPSCs but did not affect their amplitude. Ionomycin-induced release required extracellular Ca2+ and was prevented by pretreatment with botulinum neurotoxin serotype F, like evoked synaptic transmission. Unlike evoked transmission, however, this increase did not involve activation of voltage-dependent Ca2+ channels because it was insensitive to Cd2+. 3. Both the lanthanide gadolinium and alpha-latrotoxin produced increases in the frequency of mEPSCs and mIPSCs, but their actions were independent of extracellular Ca2+. 4. Adenosine, the gamma-aminobutyric acid-B (GABAB) receptor agonist baclofen, and a mu-opioid receptor agonist strongly reduced the frequency of synaptic currents triggered by all three secretagogues. 5. We conclude that activation of these presynaptic receptors can reduce high frequencies of vesicular glutamate and GABA release by directly impairing transmitter exocytosis. Presynaptic inhibition of gadolinium- and alpha-latrotoxin-induced release indicates that this impairment occurs without changes in intraterminal Ca2+ homeostasis and when vesicle fusion is rendered Ca2+ independent, respectively. 6. The inhibition of ionomycin-induced release provides additional evidence for a direct, neurotransmitter receptor-mediated modulation of the proteins underlying vesicular docking or fusion as an important component of presynaptic inhibition of evoked synaptic transmission.
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PMID:Presynaptic inhibition of calcium-dependent and -independent release elicited with ionomycin, gadolinium, and alpha-latrotoxin in the hippocampus. 873

The total Ca(2+)-dependent release of glutamate induced by depolarization of cerebrocortical nerve terminals with KCl was analyzed into a fast and a slow component. The fast component exhibited a decay time of < 1 s and accounted for 0.95 +/- 0.10 nmol of glutamate, whereas the slow component, which exhibited a decay time of 52 +/- 7 s, accounted for the release of 2.48 +/- 0.19 nmol of glutamate. These two components were differentially affected by the Ca2+ chelator BAPTA, the divalent cation Sr2+, or the botulinum neurotoxin A. The adenosine A1 receptor agonist N6-cyclohexyladenosine strongly reduced the fast component without altering the slow component. In contrast, the inhibitory effect of arachidonic acid and the facilitatory action of the metabotropic glutamate receptor agonist (1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid were observed as a decrease and an increase, respectively, in the two components. It is concluded, first, that the fast and slow components correspond to the release of docked and mobilized vesicles, respectively, and second, that presynaptic modulation more significantly alters the fast component of release.
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PMID:Two components of glutamate exocytosis differentially affected by presynaptic modulation. 893 66

Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca2+ ionophore) in the presence of Ca2+ and was proportional to the amount of L-glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L- or N-type voltage-gated Ca2+ channel blockers. Bay K 8644, an agonist for the L-type Ca2+ channel, stimulated the release of aspartate 2.1-fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the L form, although pinealocytes also contain the D form in their cytoplasm, corresponding to approximately 30% of the total free aspartate. Radiolabeled L-aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na+-dependent manner. The Na+-dependent uptake of L-aspartate was strongly inhibited by L-cysteine sulfinate, beta-hydroxyaspartate, and L-serine-O-sulfate, inhibitors for the Na+-dependent glutamate/aspartate transporter on the plasma membrane. Na+-dependent sequestration of L-aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that L-aspartate is released through microvesicle-mediated exocytosis from pinealocytes and is taken up again through the Na+-dependent transporter at the plasma membrane. The possible role of L-aspartate as an intercellular chemical transmitter in the pineal gland is discussed.
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PMID:L-aspartate but not the D form is secreted through microvesicle-mediated exocytosis and is sequestered through Na+-dependent transporter in rat pinealocytes. 920 28

Tricyclic antidepressants (e.g., imipramine, desipramine) are currently used in the treatment of mood disorders such as depression. At the cellular level they inhibit the re-uptake of the exocytosed monoamines serotonin and noradrenaline. However, they also stimulate phospholipase C activity and the production of the second messenger inositol 1,4,5-trisphosphate. Since phospholipase C activation can also lead to the production of the protein kinase C activator diacylglycerol, we have undertaken experiments to see whether acutely applied desipramine could change the synaptic strength of neurons in a protein kinase C-dependent manner. Experiments performed with cultured hippocampal neurons dissociated from neonatal rats revealed that desipramine rapidly enhanced the spontaneous vesicular release of glutamate. This was observed by measuring the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. Analysis of amplitude distribution histograms indicated a presynaptic site of action. The protein kinase inhibitor staurosporine and down-regulation of protein kinase C activity greatly reduced the desipramine-dependent enhancement of the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. This presynaptic modulation requires SNARE proteins because cleavage of SNAP-25 with the botulinum neurotoxin A strongly reduced the desipramine-induced glutamate release. Thus, acute applications of desipramine stimulated the ongoing neurotransmitter release pathway, probably by activating protein kinase C. Our data indicate that tricyclic antidepressant drugs not only act on serotoninergic and/or noradrenergic cells but can also modify the activity of glutamatergic neurons.
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PMID:Acute application of the tricyclic antidepressant desipramine presynaptically stimulates the exocytosis of glutamate in the hippocampus. 1021 74

1. The aim of the present study is to characterize the role of the P2X receptor in spinal nociceptive processing in vivo. We investigated the mechanisms of the P2X receptor agonist alpha,beta-methylene ATP (alpha,betameATP)-induced modulation of acute nociceptive signalling in mouse spinal cord. 2. Intrathecal administration of alpha,betameATP produced a significant and dose-dependent thermal hyperalgesic response. This response was completely blocked by intrathecal pretreatment with the non-selective P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and the selective P2X1, P2X3 and P2X2-3 receptor antagonist, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). Pretreatment with alpha,betameATP 15, 30 and 60 min prior to administration of a second dose of alpha,betameATP diminished the alpha,betameATP-induced thermal hyperalgesia. 3. A potent agonist for the P2X1 receptor, beta,gamma-methylene-L-ATP, did not show the hyperalgesic response, indicating that the P2X1 receptor is not involved in the spinal nociceptive pathway. 4. In fura-2 experiments using mouse dorsal root ganglion (DRG) neurons, alpha,betameATP (100 microM) increased intracellular Ca2+ ([Ca2+]i). This was not produced by a second application of alpha,betameATP. The same DRG neurons also showed a marked [Ca2+]i increase in response to capsaicin (3 microM). 5. Intrathecal pretreatment with the Ca2+-dependent exocytosis inhibitor, botulinum neurotoxin B, abolished the thermal hyperalgesia by alpha,betameATP. Furthermore, thermal hyperalgesia was significantly inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonists, 2-amino-5-phosphonopentanoate (APV), dizocilpine and ifenprodil. 6. These findings suggest that alpha,betameATP-induced thermal hyperalgesia may be mediated by the spinal P2X3 receptor subtype that causes unresponsiveness by repetitive agonist applications, and that alpha,betameATP (perhaps through P2X3 receptors) may evoke spinal glutamate release which, in turn, leads to the generation of thermal hyperalgesia via activation of NMDA receptors.
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PMID:In vivo pathway of thermal hyperalgesia by intrathecal administration of alpha,beta-methylene ATP in mouse spinal cord: involvement of the glutamate-NMDA receptor system. 1038 45

Type A botulinum neurotoxin (BoNT/A) is a zinc endopeptidase that contains the consensus sequence HEXXH (residues 223-227) in the toxic light chain (LC). The X-ray structure of the toxin has predicted that the two histidines of this motif are two of the three zinc-coordinating ligands and that the glutamate is a crucial amino acid involved in catalysis. The functional implication of E224 in the motif of LC was investigated by replacing the residue with glutamine and aspartate using site-directed mutagenesis. Substitution of Glu-224 with Gln (E224Q) resulted in a total loss of the endopeptidase activity, whereas substitution with Asp (E224D) retained about 1.4% of the enzymatic activity (k(cat) 140 vs 1.9 min(-1), respectively). However, K(m) values for wild-type and E224D BoNT/A LC were similar, 42 and 50 microM, respectively. Global structure, in terms of secondary structure content and topography of aromatic amino residues, Zn(2+) content, and substrate binding ability are retained in the enzymatically inactive mutants. Titration of Zn(2+) to EDTA-treated wild-type and mutant proteins indicated identical enthalpy for Zn(2+) binding. These results suggest an essential and direct role of the carboxyl group of Glu-224 in the hydrolysis of the substrate. The location of the carboxyl group at a precise position is critical for the enzymatic activity, as replacement of Glu-224 with Asp resulted in almost total loss of the activity.
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PMID:Probing the mechanistic role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain. 1069 9


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