Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies have shown that polychlorinated biphenyls may affect cognitive functions both in human and also in experimental animals. One of the neurochemical parameters that is changed after exposure to these compounds is a reduction in the dopamine level in the brain, although the mechanism behind this reduction is not known. We have therefore investigated whether this reduction could be caused by an effect on vesicular uptake. ortho-Chlorinated biphenyls are found to be competitive inhibitors of dopamine transport into synaptic vesicles from rat brain with K(i) concentrations as low as 4 microM. In contrast, several nonortho-chlorinated biphenyls did not inhibit vesicular uptake. The inhibition was specific for dopamine, in that the uptake of glutamate and GABA was inhibited at higher PCB concentrations under identical conditions. The vesicular Mg-ATPase proton pump was also inhibited at higher concentrations of PCBs than the dopamine transport. Uptake of methylamine gave no indication of any disruption of the vesicular proton gradient. The inhibition of dopamine vesicular uptake by PCBs was competitive. Several of the ortho-PCBs also inhibited the binding of tetrabenazine, which is known to bind to a site close to the dopamine binding site, at the vesicular transporter. The results show that inhibition of vesicular uptake may contribute to the decrease of dopamine reported in nervous tissue after exposure to PCBs under different conditions.
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PMID:The effect of polychlorinated biphenyls on the uptake of dopamine and other neurotransmitters into rat brain synaptic vesicles. 1062 Apr 85

The consequence of reduced uptake of neurotransmitters into synaptic vesicles on synaptic transmission was examined in rat hippocampal slices and culture using bafilomycin A1 (Baf), a potent and specific blocker of the vacuolar-type (V-type) ATPase, which eliminates the driving force for the uptake of both glutamate and GABA into synaptic vesicles. After incubation with Baf, both the amplitude and frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were reduced in the slice preparation. Similar effects were seen with glutamatergic miniature excitatory postsynaptic currents (mEPSCs) and GABAergic mIPSCs from cultured neurons. This result indicates that vesicular content is reduced by Baf. The dramatic reduction in the frequency of mPSCs could result either from the exocytosis of empty vesicles or from a mechanism which prevents the exocytosis of depleted vesicles. Vesicle cycling was directly examined using confocal imaging with FM 1-43. In the presence of Baf, vesicles could still be endocytosed and they were released at the same probability as from control untreated synapses. Prolonged high-frequency electrical stimulation of synapses in culture failed to alter the amplitude of mEPSCs, suggesting that the filling of vesicles is rapid compared to the rate of vesicle recycling during repetitive synaptic stimulation. Profound release of glutamate with alpha-latrotoxin did cause a small, but reproducible, reduction in quantal size. These results indicate that decreasing the amount of glutamate and GABA in synaptic vesicles reduces quantal size. Furthermore, the probability of vesicle exocytosis appears to be entirely independent of the state of filling of the vesicle. However, even during high-frequency action potential-evoked release of glutamate, quantal size remained unchanged.
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PMID:Effects of reduced vesicular filling on synaptic transmission in rat hippocampal neurones. 1081 37

Classical (non-peptide) transmitters are stored into secretory vesicles by a secondary active transporter driven by a V-type H(+)-ATPase. Five vesicular neurotransmitter uptake activities have been characterized in vitro and, for three of them, the transporters involved have been identified at the molecular level using cDNA cloning and/or Caenorhabditis elegans genetics. These transporters belong to two protein families, which are both unrelated to the Na(+)-coupled neurotransmitter transporters operating at the plasma membrane. The two isoforms of the mammalian vesicular monoamine transporter, VMAT1 and VMAT2, are related to the vesicular acetylcholine transporter (VACHT), while a novel, unrelated vesicular inhibitory amino acid transporter (VIAAT), also designated vesicular GABA transporter (VGAT), is responsible for the storage of GABA, glycine or, at some synapses, both amino acids into synaptic vesicles. The observed effects of experimentally altered levels of VACHT or VMAT2 on synaptic transmission and behavior, as well as the recent awareness that GABAergic or glutamatergic receptors are not always saturated at central synapses, suggest a potential role of vesicular loading in synaptic plasticity.
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PMID:The loading of neurotransmitters into synaptic vesicles. 1086 21

Extracts of St. John's Wort are widely used for the treatment of depressive disorders. The active principles have not yet been finally elucidated. We have recently shown that hyperforin, a major active constituent of St. John's Wort, not only inhibits the neuronal uptake of serotonin, norepinephrine and dopamine, but also that of L-glutamate and GABA. No other antidepressant compound exhibits a similar broad uptake inhibiting profile. To investigate this unique kind of property, kinetic analyses were performed regarding the uptake of 3H-L-glutamate and 3H-GABA into synaptosomal preparations of mouse brain. Michaelis-Menten kinetics revealed a reduction of Vmax (8.27 to 1.80 pmol/mg/min for 3H-L-glutamate, 2.76 to 0.77 pmol/mg/min for 3H-GABA) while Km was nearly unchanged in both cases, suggesting non-competitive inhibition. The unselective uptake inhibition by hyperforin could be mimicked by the Na+-ionophore monensin and by the Na+-K+-ATPase inhibitor ouabain. However, both mechanisms can be discarded for hyperforin. Several amiloride derivatives known to affect sodium conductance significantly enhance 3H-GABA and 3H-L-glutamate uptake and inhibit the uptake inhibition by hyperforin, while monensin or ouabain inhibition were not influenced. Selective concentrations of benzamil for amiloride sensitive Na+-channels and selective concentrations of 5'-ethylisopropylamiloride (EIPA) for the Na+-H+-exchangers both had an attenuating effect on the hyperforin inhibition of L-glutamate uptake, suggesting a possible role of amiloride sensitive Na+-channels and Na+-H+-exchangers in the mechanism of action of hyperforin.
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PMID:Inhibition of synaptosomal uptake of 3H-L-glutamate and 3H-GABA by hyperforin, a major constituent of St. John's Wort: the role of amiloride sensitive sodium conductive pathways. 1088 45

To facilitate the discovery of novel compounds that modulate human GABA(A) receptor function, we have developed a high throughput functional assay using a fluorescence imaging system. L(tk-) cells expressing combinations of human GABA(A) receptor subunits were incubated with the pH-sensitive dye 2',7'bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, then washed and placed in a 96-well real-time fluorescence plate reader. In buffer adjusted to pH 6.9 there was a robust and persisting acidification response to addition of GABA, which was antagonised by the GABA(A) receptor antagonist bicuculline. The concentration-response relationship for GABA was modulated by allosteric ligands, including benzodiazepine (BZ) site agonists and inverse agonists. The effects of BZ site ligands on the pH response to GABA for receptors containing alpha1beta3gamma2, alpha3beta3gamma2 or alpha5beta3gamma2 subunits were well correlated with results from electrophysiological studies on the same receptor subunit combinations expressed in Xenopus oocytes. Most modulatory compounds tested were found to be relatively unselective across the three subunit combinations tested; however, some showed subtype-dependent efficacy, such as diazepam, which had highest agonist effects on the alpha3beta3gamma2 subtype, substantial but lesser agonism on alpha1beta3gamma2 and still substantial but the least agonism on alpha5beta3gamma2. This indicates that the alpha subunit within the recombinant receptor expressed in L(tk-) cells can affect the efficacy of the response to some BZ compounds. Inhibitors of Na(+)/Cl(-) cotransport, anion/anion exchange and the gastric type of H(+)/K(+) ATPase potently inhibited GABA-evoked acidification, indicating that multiple transporters are involved in the GABA-evoked pH change. This novel fluorescence-based high throughput functional assay allows the rapid characterization of allosteric ligands acting on human GABA(A) receptors.
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PMID:Pharmacology of recombinant human GABA(A) receptor subtypes measured using a novel pH-based high-throughput functional efficacy assay. 1093 48

At the synapse, neurotransmitter release is triggered physiologically by Ca(2+) influx through voltage-gated Ca(2+) channels. Non-physiologically, release can be evoked by a potent neurotoxin, alpha-latrotoxin, and by hypertonic sucrose. Controversy has arisen on whether release evoked by alpha-latrotoxin and hypertonic sucrose requires extracellular Ca(2+) or Ca(2+) from intracellular stores. Using synaptosomes, we have studied the Ca(2+) dependence of alpha-latrotoxin and sucrose action in different neurotransmitter systems. In agreement with previous data, no requirement for extracellular Ca(2+) in sucrose-induced secretion of norepinephrine, dopamine, glutamate or GABA was detected. Unexpectedly, we observed large differences between these neurotransmitters in the Ca(2+) dependence of alpha-latrotoxin-stimulated release: norepinephrine release required Ca(2+), dopamine release was only partially Ca(2+) dependent, and glutamate and GABA release did not require Ca(2+). To test if Ca(2+) derived from intracellular Ca(2+) stores participates in neurotransmitter release triggered by alpha-latrotoxin or hypertonic sucrose, we employed thapsigargin, a Ca(2+)-ATPase inhibitor that empties Ca(2+) stores. Thapsigargin did not induce neurotransmitter release, nor did it inhibit subsequent release stimulated by KCl depolarization, hypertonic sucrose or alpha-latrotoxin. However, intracellular Ca(2+) performs an important regulatory function, since thapsigargin increased the size of the readily releasable pool as measured by stimulation with hypertonic sucrose. This effect required extracellular Ca(2+) and protein kinase C, suggesting that depletion of internal Ca(2+) stores leads to store-operated Ca(2+) entry. The resulting Ca(2+) influx does not trigger release by itself, but activates protein kinase C that increases the readily releasable pool of neurotransmitters. Our data show that internal and external Ca(2+) is not acutely involved in hypertonic sucrose-evoked neurotransmitter release, while alpha-latrotoxin-triggered release requires external Ca(2+) for a subset of neurotransmitters. Although internal Ca(2+) is not essential for release, it modulates its extent, implying that the emptying of intracellular stores by activation of presynaptic receptors plays an important regulatory role in neurotransmitter release.
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PMID:Role of calcium in neurotransmitter release evoked by alpha-latrotoxin or hypertonic sucrose. 1111 28

The ability of a series of specific Galpha carboxyl-terminal antisera, (i.e. anti-Gsalpha, anti-Gi1/2alpha, anti-Gi3alpha/Goalpha, anti-Goalpha/Gi3alpha, and anti-Gq/11alpha) to disrupt (+/-)-baclofen-stimulated high-affinity guanosine triphosphatase (GTPase) activity was explored in rat cerebral cortical membranes to identify the Galpha subunit(s) involved in gamma-aminobutyric acid(B) (GABA(B)) receptor-mediated signal transduction. Pretreatment of the membranes with the AS/7 (anti-Gi1/2alpha) antiserum inhibited GABA(B) receptor-mediated response without affecting the basal activity. The RM/1 (anti-Gsalpha) and QL (anti-Gq/11alpha) antisera failed to inhibit GABA(B) receptor-coupled responses. The results of the EC/2 (anti-Gi3alpha/Goalpha) and GO/1 (anti-Goalpha/Gi3alpha) antisera were difficult to interpret since the basal activities were influenced by these antisera. These results, in conjunction with the data in our previous reconstitution study, indicate that Gi2alpha is a main transducer of GABA(B) receptor-mediated signaling in rat cerebral cortex.
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PMID:Identification of galpha subtype(s) involved in gamma-aminobutyric acid(B) receptor-mediated high-affinity guanosine triphosphatase activity in rat cerebral cortical membranes. 1112 89

We identified two mammalian ULK1 (Unc-51-like kinase involved in neurite extension) binding proteins by yeast two-hybrid screening. Both proteins showed high structural similarity to microtubule-associated protein (MAP) light chain 3 (LC3). One is identical to the Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), an essential factor for intra-Golgi transport [39]. The other is identical to the gamma 2-subunit of GABA-A receptor associated protein (GABARAP) which has a possible role in receptor transport [46]. Using the yeast two-hybrid system and the in vitro GST pull-down assay, we found that the N-terminal proline/serine rich (PS) domain of ULK1 (amino acid 287-416) is required for ULK1-GATE-16 and ULK1-GABARAP protein interactions. However, the kinase activity of ULK1 affected neither ULK1-GATE-16 nor ULK1-GABARAP interaction. Immunohistochemical analysis using ULK1 and GABARAP antibodies showed that the ULK1 and the GABARAP proteins co-localized to many kind of neurons such as pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, and Purkinje cells of the cerebellum. In HeLa cells, endogenous ULK1 and tagged GABARAP showed punctate structures in the cytosol, and were colocalized. These results suggest that the interaction of ULK1 and GABARAP is important to vesicle transport and axonal elongation in mammalian neurons.
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PMID:Interaction of the Unc-51-like kinase and microtubule-associated protein light chain 3 related proteins in the brain: possible role of vesicular transport in axonal elongation. 1114 1

Synaptic vesicles in the nerve terminal play a pivotal role in neurotransmission. Neurotransmitter accumulation into synaptic vesicles is catalyzed by distinct vesicular transporters, harnessing an electrochemical proton gradient generated by V-type proton-pump ATPase. However, little is known about regulation of the transmitter pool size, particularly in regard to amino acid neurotransmitters. We previously provided evidence for the existence of a potent endogenous inhibitory protein factor (IPF), which causes reduction of glutamate and GABA accumulation into isolated, purified synaptic vesicles. In this study we demonstrate that IPF is concentrated most in the synaptosomal cytosol fraction and that, when introduced into the synaptosome, it leads to a decrease in calcium-dependent exocytotic (but not calcium-independent) release of glutamate in a concentration-dependent manner. In contrast, alpha-fodrin (non-erythroid spectrin), which is structurally related to IPF and thought to serve as the precursor for IPF, is devoid of such inhibitory activity. Intrasynaptosomal IPF also caused reduction in exocytotic release of GABA and the monoamine neurotransmitter serotonin. Whether IPF affects vesicular storage of multiple neurotransmitters in vivo would depend upon the localization of IPF. These results raise the possibility that IPF may modulate synaptic transmission by acting as a quantal size regulator of one or more neurotransmitters.
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PMID:IPF, a vesicular uptake inhibitory protein factor, can reduce the Ca(2+)-dependent, evoked release of glutamate, GABA and serotonin. 1118 35

It is widely accepted that energy deprivation causes a neuronal death that is mainly determined by an increase in the extracellular level of glutamate. Consequently an excessive membrane depolarization and a rise in the intracellular concentration of sodium and calcium are produced. In spite of this scenario, the function of excitatory and inhibitory amino acids during an episode of energy failure has not been studied yet at a cellular level. In a model of cerebral hypoglycemia in the rat substantia nigra pars compacta, we measured neuronal responses to excitatory amino acid agonists. Under single-electrode voltage-clamp mode at -60 mV, the application of the ionotropic glutamate receptor agonists N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainate, and the metabotropic group I agonist (S)-3,5-dihydroxyphenilglycine (DHPG) produced reversible inward currents in the dopaminergic cells. In addition, an outward current was caused by the superfusion of the metabotropic GABA(B) agonist baclofen. Glucose deprivation enhanced the inward responses caused by each ionotropic glutamate agonist. In contrast, hypoglycemia depressed the DHPG-induced inward current and the baclofen-induced outward current. These effects of hypoglycemia were reversible. To test whether a failure of the Na(+)/K(+) ATPase pump could account for the modification of the agonist-induced currents during hypoglycemia, we treated the midbrain slices with strophanthidin (1-3 microM). Strophanthidin enhanced the inward currents caused by glutamate agonists. However, it did not modify the GABA(B)-induced outward current. Our data suggest that glucose deprivation enhances the inward current caused by the stimulation of ionotropic glutamate receptors while it dampens the responses caused by the activation of metabotropic receptors. Thus a substantial component of the augmented neuronal response to glutamate, during energy deprivation, is very likely due to the failure of Na(+) and Ca(2+) extrusion and might ultimately favor excitotoxic processes in the dopaminergic cells.
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PMID:Hypoglycemia enhances ionotropic but reduces metabotropic glutamate responses in substantia nigra dopaminergic neurons. 1124 85


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