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1.
A study was made of the time course and kinetics of [3H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [3H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent Km values for GABA neurons for high and low affinity uptake were 0.33 × 10−6 M and 41.8 × 10−4 M, respectively. For nonneuronal cells, the apparent Km for high affinity uptake was 0.29 × 10−6 M. The apparent Vmax values for GABA neurons for high and low affinity uptake were 28.7 × 10−6 mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent Vmax for high affinity uptake was 0.06 × 10−6 mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10−9 M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [3H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [3H]GABA (4.4 × 10−6 M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.  相似文献   

2.
The principal sites of γ-aminobutyric acid (GABA) uptake in lobster nerve-muscle preparations have been determined with radioautographic techniques after binding of the amino acid to proteins by aldehyde fixation. Semiquantitative studies showed that about 30% of the radioactive GABA taken into the tissue was bound to protein by fixation. Both light and electron micrographs showed dense accumulations of label over Schwann and connective tissue cell cytoplasm; muscle was lightly labeled, but axons and terminals were almost devoid of label. The possible role of Schwann and connective tissue cells in the inactivation of GABA released from inhibitory axons is discussed.  相似文献   

3.
Changes in the effective membrane resistance of the abductor muscle of the dactylopodite of the crayfish were used to indicate changes in the GABA concentration in the synaptic cleft. Following bath application of GABA (10?5 to 5 × 10?5M), the muscle membrane resistance decreased and then increased slowly over the next few minutes. Renewing the solution or stirring the bath restored the GABA effect. Higher GABA concentrations produced a large stable decrease in membrane resistance. An active uptake system for GABA in the junctional region is suggested by the observation that the slow increase in membrane resistance following GABA application was decreased by cooling to 2°C or by the addition of known GABA uptake blockers such as L -DABA, β-guanidinopropionic acid, or nipecotic acid. The transport inhibitors, PCMBS and chlorpromazine, produced irreversible decreases in muscle membrane resistance, which precluded examining their effects on GABA inactivation. The decrease in GABA effect was not dependent on the external sodium concentration or on the degree of receptor activation. Nipecotic acid, which blocked GABA inactivation, did not affect the decay of the neurally evoked inhibitory junctional potential.  相似文献   

4.
The effect of thiopental on rat brain hippocampal GABA receptors was studied in slice preparations and membrane fractions. In slice preparations, thiopental at a concentration of 10−5 M enhanced the GABA (1−5 × 10−4M) inhibition of the field potentials evoked in pyramidal neurons by stratum radiatum stimulation. In hippocampal slices obtained from chronically barbital-administered (100 mg/kg, b.i.d., 10 days) rats, less enhancement of thiopental on GABA inhibition of the field potentials was observed. In binding experiments, two components of specific [3H]GABA binding to membrane fractions were observed. While thiopental was without effect on high-affinity sites, [3H]GABA binding with low affinity was enhanced by 80% in the presence of 10−5 M thiopental. The results are discussed in relation to the phenomena underlying chronic barbiturate administration.  相似文献   

5.
—A rapid accumulation of [3H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [3H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [3H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated Km value for GABA is 2·2 × 10?5m , and Vmax is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [3H]GABA when cortical slices are exposed to a GABA-free medium. [3H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.  相似文献   

6.
The effects of membrane lipid disturbances induced by ischemia and exogenously added lipids on the uptake of GABA and Ca2+ were investigated in gerbil brain synaptosomes. Ischemia was produced by bilateral ligation of common carotid arteries in Mongolian gerbil for 10 min. The level of the free fatty acids (FFA) increased significantly in ischemic synaptosomes. Incorporation of [1-14C]arachidonate into membrane phosphatidylinositol and phosphatidylcholine was decreased by about 20–35%. Furthermore ischemia exerted an inhibitory effect on GABA uptake but remained without effect on calcium accumulation. Thiopental application in dose of 100 mg per kg body weight 30 min before ischemia caused a protective effect on membrane lipid disturbances induced by ischemia and enhanced GABA uptake. Unsaturated fatty acids (arachidonate and docosahexanoate) in concentration of 10−5−10−4 mol/l and lysocompounds (lysophosphatidylcholine and lysophosphatidylethanolamine) in concentrations higher than 10−4 mol/l decreased GABA and Ca2+ uptake in synaptosomes from normoxic brains. No effect was seen with saturated stearic acid. These results suggest that the inhibition of GABA uptake into ischemic synaptosomes resulted from an action of unsaturated fatty acids, arachidonic and docosahexanoic acids which were liberated during ischemia. Moreover the transient higher local concentration of lysophospholipids close to GABA carrier system may also have contributed to the inhibition observed during ischemia.  相似文献   

7.
Isoguvacine Binding, Uptake, and Release: Relation to the GABA System   总被引:4,自引:2,他引:2  
Isoguvacine (1,2,3,6-tetrahydropyridine-4-car-boxylic acid) is a GABA (γ-aminobutyric acid) agonist with limited conformational flexibility. In these studies we investigated the binding, uptake, and release of [3H] isoguvacine by use of tissue preparations of rat CNS, comparing the results with similar studies of [3H]GABA. The results from these investigations indicate that isoguvacine binds to membrane preparations of rat forebrain with pharmacological characteristics similar to the post-synaptic GABA recognition site; that it is transported into synaptosomal preparations by an uptake system similar to the high-affinity GABA uptake system; and that recently accumulated isoguvacine is released in a Ca2+-dependent manner and by heteroexchange with external GABA. The ability of isoguvacine and γ-hydroxybutyric acid to decrease the K+-stimulated Ca2+-dependent release process was also investigated. The results indicate that isoguvacine interactions have many of the biochemical features of GABA synaptic function, isoguvacine being, however, less potent than GABA.  相似文献   

8.
It was found that rat brain nerve endings contain a high affinity and Na- dependent transport system for [3H]β-alanine ([3H]β-ala). As determined from Michaelis-Menten plots, the [3H]β-ala Km was 2.8 × 10-5 M and the Vmax was 0.29 nmol/mg protein/5 min. Under similar incubation conditions the [3H]GABA Km was 3.8 x 10-6M and the Vmax was 6.3 nmol/mg protein/5 min. The [3H]β-ala and [3H]GABA transport systems were further characterized by determining the IC50 values for a number of compounds. The compounds tested were GABA, β-ala, l -2,4-diaminobutyric acid. DL-3-hyd-roxy-GABA, β-guanidopropionic acid, strychnine, γ-guanidobutyric acid, imidazole-4-acetic acid, DL-proline, bicuculline, L-serine, glycine, l -α-ala and taurine. DABA, dl -3-hydroxy-GABA, β-guanidopro-pionic acid and γ-guanidobutyric acid were more potent inhibitors of [3H]GABA than [3H]β-ala transport. Strychnine, imidazole-4-acetic acid, proline and glycine were between 2 and 6 times more potent inhibitors of [3H]β-ala than [3H]GABA transport. β-Ala, bicuculline, serine, α-alanine and taurine were all markedly more potent (12–150 times) inhibitors of [3H]β-ala than [3H]GABA transport. IC50 values were also determined for the above compounds for the sodium-dependent and the sodium-independent binding of [3H]GABA to both fresh and frozen brain membranes. In general, the potency of these compounds to inhibit either sodium-independent or sodium-dependent binding was greater in fresh tissue. It was also observed that the neurophysiologically‘glycine-like’amino acids were more potent inhibitors in the presence of NaCl. No significant correlations were found between [3H]GABA binding under any condition and [3H]GABA or [3H]β-ala transport into nerve endings.  相似文献   

9.
Abstract— The glutamic acid decarboxylase has been purified from the lobster central nervous system. Potassium ion (0-075 m ) and β-mercaptoethanol (0-025 m ) were essential for enzyme activity. Enzyme had about 60 per cent of its optimal activity in the absence of added pyridoxal phosphate. Carbonyl reagents (10?4m -hydroxylamine or amino oxyacetic acid) would abolish this residual activity. The pH optimum of the enzyme was about 8-0. Standard Michaelis-Menten kinetics were applied to the decarboxylation of glutamate and a Km of 0.02 m was calculated. GABA inhibited the reaction (Ki= 1.25 × 10?3m ), but the inhibition showed anomalous behaviour when graphed by the method of Lineweaver and Burk (1934). The GABA inhibition resembled competitive inhibition, but curves rather than straight lines intersecting at a common point on the velocity axis were obtained. This effect remains unexplained. Preliminary studies failed to reveal any subunit structure of the enzyme. The sedimentation coefficient (.S20.w) was 6-55 in a sucrose density gradient in an ultracentrifuge. This was unchanged by the addition of any of the agents that influence enzyme activity. The subcellular localization of the decarboxylase was explored in crude homogenates of lobster central nervous system prepared in various ways. The major proportion (about 90 per cent) of the enzyme activity was in the soluble fraction.‘Particulate’enzyme could be prepared, but gentle suspension of this material in buffer liberated most of the activity. A contaminant in the radioactive substrates led to the production of radioactive GABA without the simultaneous evolution of CO2. In this case, GABA production required active enzyme but was not an exclusive property of the glutamic decarboxylase activity.  相似文献   

10.
The active uptake of [3H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [14C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [3H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [14C]GABA (25.8–74.1). GABA, 10–4 M, and pipecolic acid, 10–4 M, produced a significant inhibition of [3H]pipecolic acid uptake into P2 fractions. Pipecolic acid, 10–4 M, significantly reduced the synaptosomal and glial uptake of [14C]GABA. GABA, 10–4 M, affected neither spontaneous nor high K+-induced release of [3H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.  相似文献   

11.
《Process Biochemistry》2004,39(5):633-636
The influence of tricyclazole on the production of phenolic precursors, glucose uptake and production of fungal mass of Fonsecaea pedrosoi was assessed in the presence of phenylalanine, sodium nitrate and tryptophan. Photocolorimetry was used for the quantitation of glucose and total phenol, and the dry weight method for biomass. The glucose uptake with phenylalanine was 99.85×103 mg/ml; with tryptophan, 99.88×103 mg/ml and with sodium nitrate, 99.90×103 mg/ml. Production of biomass: with tryptophan, 2.7×10−1 mg; with sodium nitrate, 3.0×10−1 mg and with phenylalanine, 3.4×10−1 mg. Tricyclazole induced higher phenol accumulation, lower glucose uptake, inhibition of melanin deposition on the cell and higher production and higher biomass production in relation to glucose uptake for all nitrogen sources tested under these conditions. A negative correlation between biomass production and glucose uptake was observed in the presence of secondary metabolism.  相似文献   

12.
The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS. The [3H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABAA and GABAB receptors. The most potent inhibitors of [3H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [35S]TBPS was partially inhibited by 1.0 × 10−6 M GABA, whilst binding of [3H]flunitrazepam was enhanced by 1.0 × 10−7 M GABA. The pharmacological profile of the [3H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS.  相似文献   

13.
Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na+, K+, and H+ on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na+ dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na+/GABA symporter energized by Na+‐exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.  相似文献   

14.
THE UPTAKE OF GABA INTO RAT SPINAL ROOTS   总被引:1,自引:0,他引:1  
—Dorsal and ventral roots, dissected from rats anaesthetized with sodium pentobarbitone, accumulated three to four times more GABA than l -glutamate, 1-aspartate or glycine during 30 min incubation at 37°C. GABA was taken up into spinal roots by a structurally specific, sodium-dependent process with an apparent Km of approx. 3 × 10?5m . This uptake process appears to be very similar to that described in rat brain, spinal cord and dorsal root ganglia.  相似文献   

15.
Glomerular particles were isolated from the bovine cerebellar vermis and studied in vitro to further assess the possibility that -aminobutyric acid (GABA) is utilized as a neurotransmitter in this synaptic complex. Cerebellar glomeruli accumulated [3H]GABA at two different high affinity sites, with affinities (K T) of 2.2×10–6M and 3.0×10–5M. These uptake sites could not be distinguished on the basis of their temperature sensitivities, sodium dependence, substrate specificities or responses to metabolic inhibitors. Although an exchange process contributed to the uptake measured in these experiments, a considerable amount of the [3H]GABA accumulated by glomerular particles was stored in an osmotically-sensitive, nonexchangeable pool. Glomerular particles preloaded with [3H]GABA exhibited a Ca2+-independent release of this amino acid in response to membrane depolarization. However, when preloaded glomerular particles were exposed to unlabeled GABA, which presumably displaced [3H]GABA from the exchangeable pool, a K-evoked and Ca2+-dependent release of the remaining [3H]GABA occurred. The observed net uptake, together with the depolarization-induced and Ca2+-dependent release, of [3H]GABA from glomerular particles supports the suggestion that functionally active GABAergic synapses are present in these structures.  相似文献   

16.
High-affinity uptake of [3H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V max) and, to a smaller extent, theK m of [3H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [3H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [3H]GABA, [3H]ACHC, or [3H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.  相似文献   

17.
The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK m andV max for GABA uptake, with an increase in theV max/K m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na+, K+-ATPase acting as an ion pump.  相似文献   

18.
The possible existence of endogenous substances other than γ-aminobutyric acid (GABA), that can also bind to rat brain GABA receptors, has been investigated in synaptic membranes derived from whole rat brain, or from cerebral cortex; as well as in isolated synaptic vesicles obtained from cerebral cortex, striatum, hypothalamus, cerebellum and spinal cord and in the superfusion fluid of electrically stimulated brain cortex slices, where a GABA-like substance is released by a calcium-dependent process. The detector used to study the presence of such presumed non-GABA endogenous ligands, were frozen and thawed rat brain synaptic membranes, that had been treated with 0.05% Triton X-100 and thoroughly washed. With this highly sensitive preparation, at least 5 pmol of GABA/ml could be detected. The extracts of the different preparations where these hypothetical ligands were looked for, were analyzed by means of gel filtration on Sephadez G-10, paper chromatography and high voltage electrophoresis. In a very great number of experiments performed, the only endogenous ligand detected was GABA itself.The possible influence of a number of peptides on binding of GABA to its receptor, was also looked for. No significant effect was found for substance P, neurotensin, cholecystokinin octapeptide sulfated, somatostatin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, methionine enkephalin (all 10?5 M), angiotensin II (10?4 M), ACTH (3 × 10?7M), poly-l-lysine (30 μg/ml) or poly-l-glutamate (30 μg/ml).  相似文献   

19.
GABA transport by nerve ending fractions of cat brain   总被引:2,自引:2,他引:0  
Abstract— Nerve ending and mitochondrial fractions were prepared from cat cerebral cortex by differential and sucrose density gradient centrifugation. The isolated fractions were characterized morphologically and enzymatically and the nerve-ending fraction was shown to be particularly rich in glutamate decarboxylase activity. The uptake of [3H] γ-aminobutyric acid (GABA) was studied by trapping the particles on a Millipore filter. GABA uptake was not saturable over the concentration range studied (0.018 to 8000 μM. Tissue/medium ratios were consistently greater than 2·5, and uptake was significantly reduced by ouabain and iodoacetate, or by replacing sodium with lithium or choline chloride. GABA uptake was significantly inhibited by desmethylimipramine (DMI) with an IC50 of approx. 10–20 μM. GABA uptake was reduced by structurally similar amino acids lacking an α-amino group, and also by methionine and phenylalanine, but not by short chain neutral amino acids.  相似文献   

20.
Abstract— Several parameters of GABA Auxes across the synaptosomal membrane were studied using synaptosomes prepared from the brain of immature (8-day-old) rats. The following aspects of GABA carrier-mediated transport were similar in immature and mature synaptosomes: (1) magnitude of [3H]GABA accumulation; (2) GABA homoexchange in normal ionic conditions; (3) GABA homoexchange in the presence of cationic fluxes (Na+ and Ca2+ influx, K+ efflux) characteristic of physiological depolarization. As in adult synaptosomes (Levi & Raiteri , 1978), in these conditions the stoichiometry of GABA homoexchange was in the direction of net outward transport (efflux > influx). The essential differences between the behaviour of 8-day-old and adult synaptosomes were the following: (1) β-alanine (a glial uptake inhibitor) inhibited GABA uptake in immature synaptosomes (the inhibition being greater in crude than in purified preparations) and was without a significant effect in adult synaptosomes. DABA and ACHC (two neuronal uptake inhibitors) depressed GABA uptake more efficiently in purified than in crude immature synaptosomes, but were as effective in crude and purified nerve endings from adult animals. The data suggest a greater uptake of GABA in the‘gliosomes’contaminating the synaptosomal preparations from immature animals. (2) In immature synaptosomes prelabelled with [3H]GABA the specific radioactivity of the GABA released spontaneously or by heteroexchange (with 300 μm -OH-GABA) was the same as that present in synaptosomes, while in adult synaptosomes OH-GABA released GABA with increased specific radioactivity. The data suggest a homogeneous distribution of the [3H]GABA taken up within the endogenous GABA pool in immature, but not in mature synaptosomes. (3) In immature synaptosomes the release of GABA (radioactive and endogenous) induced by depolarization with high KC was not potentiated by Ca2+, unless the synaptosomes had been previously depleted of Na+ These data suggest that, although a Ca2+ sensitive pool of GABA may be present, this pool is not susceptible to being released in normal conditions, probably because the high intrasynaptosomal Na+ level prevents a sufficient depolarization. The possible significance of these findings in terms of functional activity of GABAergic neurotransmission in the immature brain is discussed.  相似文献   

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