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1.
THE COMPLEX BINDING OF γ-AMINOBUTYRIC ACID TO A RAT BRAIN SYNAPTIC MEMBRANE PREPARATION 总被引:1,自引:1,他引:0
The association of γ-aminobutyric acid (GABA) with rat brain synaptic membranes is complex and involves more than one set of binding sites. This fact is suggested by the form of experimentally determined binding curves and confirmed by the destruction of one set of homogenous sites by covalent modifications with iodine. The difference in binding of GABA by two sets of membranes which have been exhaustively iodinated in the presence and absence of an excess of GABA corresponds to 1/6 of the binding expressed by uniodinated membranes after equilibration with 0.25 μm -GABA. This difference appears to be the result of the expression of a homogeneous set of independent sites, each of which contains an iodinatable'residue that is protected by bound GABA. Examination of the factors leading to the retention of radioactive ligand by a synaptic membrane preparation is suggestive of a method for determining the maximum number of binding sites for a given ligand and for evaluating solution space without displacing the bound species or adding a presumably inert tracer. Three additional measurements of retention of high ligand concentration are shown to be sufficient for the determination. Employment of the method facilitates the investigation of the interaction of the membrane preparations with individual agonists and antagonists. 相似文献
2.
Abstract— Slices of rat cerebral cortex were labelled by incubation with [3 H]γ-aminobutyric acid (GABA) and homogenized in isotonic sucrose. The subcellular distributions of endogenous GAB A, [3 H]GABA and glutamate decarboxylase (GAD) were studied by density gradient centrifugation. The subcellular distributions of the labelled and endogenous amino acid were remarkably similar, indicating that [3 H]GABA is taken up into the endogenous GABA pool. About 40 per cent of both endogenous and [3 H]GABA were recovered in particles which were tentatively identified as synaptosomes from their equilibrium density and sensitivity to osmotic shock. In slices labelled with [3 H]GABA and [14 C]α-aminoisobutyric (AIB) acid, significantly more [3 H]GABA was recovered in paniculate fractions than [14 C]AIB. About 80 per cent of the enzyme GAD was also recovered in the same particle fractions which contained [3 H]GABA and endogenous GABA. Evidence is presented which suggests that a loss of particle-bound GABA occurs during subcellular fractionation procedures. 相似文献
3.
—The specific binding of [3H]kainic acid to synaptic membranes from rat brain was saturable with a dissociation constant of about 60 nm . The apparent maximal number of binding sites was about 1 pmol/mg protein. The most effective displacer of specific [3H]kainic acid binding was quisqualic acid, a powerful excitant which is structurally similar to l -glutamate. However, quisqualic acid was one-third as potent a displacer as kainic acid itself. l -Glutamate was the next potent in displacing [3H]kainic acid binding, but also was less effective (1/25) than kainic acid itself. All other compounds including suspected neurotransmitters were at least an order of magnitude lower in potency compared to l -glutamate. When various tissues and brain regions were tested for specific [3H]kainic acid binding, we found the specified binding was localized to grey matter in the brain. In studies of subcellular fractionation of the brain, we found that crude synaptosomal membrane preparations were most enriched in specific [3H]kainic acid binding. Specific [3H]kainic acid binding in various regions of the rat brain varied 5- to 6-fold. 相似文献
4.
THE EFFECT OF ELECTRICAL STIMULATION AND HIGH POTASSIUM CONCENTRATIONS ON THE EFFLUX OF [3 H] γ-AMINOBUTYRIC ACID FROM BRAIN SLICES 总被引:10,自引:0,他引:10
Abstract— Brain slices were incubated with [3 H]GABA in a medium containing aminooxyacetic acid to prevent metabolism of [3 H]GABA by GABA-glutamate transaminase. The slices, which rapidly accumulated radioactivity, were then continuously perfused and the efflux of [3 H]GABA from the tissue was measured. The spontaneous efflux of [3 H]GABA consisted of an initial rapid phase followed by a much slower release of [3 [H]GABA. After 40 min perfusion 90 per cent of the radioactivity remained in the tissue.
The slices were depolarized by electrical stimulation or by perfusion with a medium containing a high potassium concentration (40 mM). These procedures caused a striking increase in the efflux of [3 H]GABA. The increased efflux produced by potassium, but not that produced by electrical stimulation, was dependent on calcium ions in the medium. The effect of electrical stimulation on [3 H]GABA release was considerably reduced by a raised concentration (10 mM) of magnesium in the medium.
High potassium concentrations and electrical stimulation did not cause an increase in the efflux of [14 C]urea, L-[3 H]leucine or [14 C]α-amino-isobutyric acid from brain slices. These results are consistent with the suggestion that GABA may be an inhibitory transmitter in the cerebral cortex. 相似文献
The slices were depolarized by electrical stimulation or by perfusion with a medium containing a high potassium concentration (40 mM). These procedures caused a striking increase in the efflux of [
High potassium concentrations and electrical stimulation did not cause an increase in the efflux of [
5.
6.
l [3H]glutamate binding was investigated in membrane preparations derived from rat cerebellum, an area of the brain where it is likely that a high density of postsynaptic glutamate receptors occurs. Glutamate was hound specifically and, in freshly prepared membranes, was optimal under physiological conditions of pH and temperature and was associated with the synaptic membrane fraction of the cell. Specific binding occurred through a single, high-affinity process with a KD, of 744 nM and a capacity of 73 pmol/mg protein. Unlike the findings reported for GABA, the specific binding of glutamate to fresh membranes did not involve an uptake site. Comparison of the potencies of a wide range of compounds with known pharmacological activities, demonstrated that their ability to displace specific glutamate binding was consistent with specific interactions with glutamate receptors. 相似文献
7.
[3H]Mepyramine binds with high affinity to membranes from brain of human, rat, guinea-pig, rabbit and mouse with drug specificity indicating an association with histamine H1receptors. Considerable species differences occur in the affinity of [3H]mepyramine, with guinea-pig and human having 34 times greater affinity than rat, mouse or rabbit. The greater affinity of [3H]mepyramine in guinea-pig than in rat is attributable both to faster association and slower dissociation rates in guinea-pig. Species differences in affinity for H1 receptor sites occur for some antihistamines but not for others. Some tricyclic antidepressant and neuroleptic drugs are extremely potent inhibitors of [3H]mepyramine binding, exceeding in potency any H1 antihistamines examined. The tricyclic antidepressant doxepin and the neuroleptic clozapine are the most potent of all drugs examined in competing for [3H]mepyramine binding. The regional distribution of specific [3H]mepyramine binding differs considerably in the various species examined. 相似文献
8.
STRUCTURE-ACTIVITY STUDIES ON THE INHIBITION OF GABA BINDING TO RAT BRAIN MEMBRANES BY MUSCIMOL AND RELATED COMPOUNDS 总被引:17,自引:11,他引:6
Abstract— A series of compounds structurally related to muscimol (5-aminomethyl-3-isoxazolol) was tested as inhibitors of the sodium-independent binding of GABA to membranes from rat brain. Muscimol, 5-(l-aminoethyl)-3-isoxazolol, 5-(2-aminoethyl)-3-isoxazolol (homomuscimol), and the bicyclic derivative 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) were relatively potent inhibitors of GABA binding. THIP is an analogue of muscimol locked in a folded conformation. The structurally related compound 1,2,3,6-tetrahydropyridine-4-carboxylic acid (isoguvacine), a semirigid analogue of trans-4-aminocrotonic acid, was also a potent inhibitor of GABA binding. Apart from muscimol, these inhibitors of GABA binding did not influence the sodium-dependent,'high-affinity' uptake of GABA in rat brain slices, whereas the potent GABA uptake inhibitors guvacine and nipecotic acid did not influence GABA binding. The present results support previous findings that different conformational modes of GABA interact with GABA postsynaptic receptors and the neuronal GABA transport system in rat brain, and indicate that the 'active conformation' of GABA with respect to the receptors is partially folded and almost planar. Based on a comparison of the present results with previous in vivo studies the structural requirements for GABA-like activity in rat cerebral cortex and cat spinal cord seem to be somewhat different. 相似文献
9.
Abstract— The spontaneous efflux of [3 H]GABA and its metabolites from the frog retina has been studied. The efflux of radioactivity was multiphasic in the presence or absence of amino-oxyacetic acid (AOAA), an inhibitor of GABA metabolism, and was not affected by light or dark adaptation.
Strong retention of radioactivity was evident in the presence of AOAA, about 90% of the label remaining in the tissue after 4 h superfusion. Under these conditions, increases in the rate of release of radioactivity were evoked by electrical stimulation, 40 m m -potassium. unlabelled GABA (5 m m ), ouabain (5 × 10−5 m ) and the absence of calcium. The amount of [3 H]GABA released by electrical stimulation was not markedly calcium dependent, whereas the response to 40 m m -potassium was reduced by 96% in the absence of calcium. 相似文献
Strong retention of radioactivity was evident in the presence of AOAA, about 90% of the label remaining in the tissue after 4 h superfusion. Under these conditions, increases in the rate of release of radioactivity were evoked by electrical stimulation, 40 m m -potassium. unlabelled GABA (5 m m ), ouabain (5 × 10
10.
A binding assay for muscarinic cholinergic receptors has been developed using labelled dexetimide as ligand and a filtration technique. The main features of this assay are its stereospecific nature, the very high affinity of the ligand for the specific receptors sitcs and its very low affinity for non-specific binding sites. The latter point was further investigated using labelled levitimide, the inactive enantiomer. The binding was found to be neither stereospecific nor saturable and displacement by both enantiomers revealed a particular curve with a very flattened course. Kinetic experiments with [3H]dexetimide suggest the occurrence of a heterogenous population of muscarinic receptors in the rat striatum. A study of the regional distribution of muscarinic receptors in rat brain showed a high concentration in the dopaminergic areas, the cortex and the hippocampus, but practically none in the cerebellum. The subcellular distribution pattern revealed a marked enrichment of [3H]dexetimide stereospecific binding sites in the microsomal fraction of rat striatum and hippocampus. Such a distribution was not found with [3H]levitimide. All the characteristics of this binding assay make dexetimide a very appropriate ligand for labelling muscarinic receptors in vitro as well as in vivo. 相似文献
11.
P. A. Bond 《Journal of neurochemistry》1973,20(2):511-517
Abstract— Slices from various regions of rat brain, incubated at 25°C, rapidly accumulate [3 H]GABA from the surrounding medium until after 60min tissue:medium ratios as high as 300 may be achieved. Kinetic analysis has demonstrated two distinct uptake systems for GABA in all the brain regions examined. One system has a relatively high substrate affinity ( Km = 1.2 ± 10-5 M) while the other has a lower affinity ( Km = 4 ± 10-4 M). Studies at low GABA concentration (5 ± 10-8 M), as well as estimates of maximum velocities, have shown that the distribution of the high affinity uptake system is heterogeneous. Cortex, hypothala mus, midbrain and hippocampus have relatively high uptake rates while the striatum, cerebellum and pons and medulla have a lower uptake rate. Maximum velocities for the low affinity uptake system show much less regional variation.
Lithium, either added to the incubation medium or fed to rats, had no effect on the uptake of GABA by cortical slices. 相似文献
Lithium, either added to the incubation medium or fed to rats, had no effect on the uptake of GABA by cortical slices. 相似文献
12.
—The binding of [14C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [14C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [14C]GABA/μg of protein, with an apparent Kd= 30 μm . The binding is competitively inhibited by bicuculline with a Ki= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle. 相似文献
13.
Abstract— The effect of amino-oxyacetic acid on the uptake of [3 H]GABA by rat brain slices was studied. When added simultaneously with [3 H]GABA, amino-oxyacetic acid had no significant effect on [3 H]GABA uptake. However, preincubation of brain slices with amino-oxyacetic acid prior to addition of [3 H]GABA produced inhibition of uptake, which increased with longer duration of preincubation. The inhibitory effect of amino-oxyacetic acid was maximal at 2 mM concentration and concentrations sufficient to inhibit significantly GABA:glutamate transaminase (10- -6 M) had no effect on [3 H]GABA uptake. D-Cycloserine and β-hydrazino-propionic acid also inhibited [3 H]GABA uptake, but the amounts required were considerably in excess of those needed to inhibit GABA:glutamate transaminase. 4-Deoxypyridoxine inhibited [3 H]GABA uptake, whether given in vivo or in vitro , and the inhibitory effect of amino-oxyacetic acid was reversed with pyridoxine. GABA transport appears to be dependent on pyridoxal phosphate and interference with this function of the vitamin is suggested as the basis for the inhibitory effect of amino-oxyacetic acid on [3 H]GABA uptake. 相似文献
14.
—Saturation binding studies with [3H]leu-enk ([tyrosyl-3, 5-3H(N)]5leu-enkephalin) revealed the presence of high and low affinity binding sites in a paniculate fraction derived from rat striatum. The binding of [3H]leu-enk to the high affinity component (KD= 2.0 ± 0.3 nM) was sensitive to morphine and levorphanol, while the binding to the low affinity component (KD= 21 ± 2 nM) was not. Incubation of the membranes, prior to assay for 30 min at 37°C, followed by centrifugation at 27, 000 g for 20 min in order to pellet the membranes allowed the detection of a factor, present in the high speed supernatant, which caused a dose-dependent inhibition of the binding of [3H]leu-enk to the morphine-sensitive and insensitive binding components. Investigations into the nature of the morphine-insensitive binding component demonstrated that it was an artifact since it was not detectable when bound and free ligand were separated by centrifugation. Furthermore, [3H]leu-enk bound to Whatman glass fiber filters, used to collect bound ligand, in a morphine-insensitive manner, and under conditions where the binding of [3H]leu-enk to the morphine sensitive component diluted proportionally with serial dilutions of the membranes, the binding to the morphine-insensitive component did not. The factor present in the high speed supernatant did not dialyze and its effects were mimicked by either trypsin or soybean trypsin inhibitor, but not by bovine serum albumin. The apparent inhibition of the binding of [3H]leu-enk to these binding components is probably not of biological significance, but the fact that the artifactual morphine insensitive binding component of striatal membranes has been shown to decrease by 20–30% following lesions of the substantia nigra suggests that the influence of this endogenous factor must be controlled for. 相似文献
15.
SOME EFFECTS OF DIETARY VITAMIN B6 DEFICIENCY ON γ-AMINOBUTYRIC ACID METABOLISM IN DEVELOPING RAT BRAIN 总被引:3,自引:3,他引:0
Abstract— Severe vitamin B6 deficiency induced in pregnant rats during the last 2 weeks of gestation resulted in a reduction of brain weight in the new born rat. This indicates that the foetus was affected in utero . However, no significant changes were observed in other measured parameters in brains of the neonates at birth. Subjecting these neonates to vitamin B6 deficiency during lactation severely retarded the development of their body and brain weights. There is evidence to suggest that B6 deficiency also leads to increased levels of glutamic acid decarboxylase apoenzyme, although the in vivo activity of the enzyme appears to be reduced as a result of marked reduction in coenzyme saturation. The level of γ-aminobutyric acid transaminase apoenzyme was reduced. Its coenzyme saturation was also reduced, but the level of reduction was less than with the decarboxylase. The progressive increase in whole brain γ-aminobutyric acid level was also retarded by the deficiency. Five days after administration of pyridoxine hydrochloride to 2-week-old deficient neonates, whole brain γ-aminobutyric acid levels and the activities of whole brain glutamic acid decarboxylase and γ-aminobutyric acid transaminase were almost restored to normal. However, brain and body weight showed a slow recovery during the same period. It was found that in the recovering neonates both enzymes follow changes in age rather than changes in brain weight. 相似文献
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17.
SOME EFFECTS OF MONOAMINE OXIDASE INHIBITORS ON THE METABOLISM OF γ-AMINOBUTYRIC ACID IN RAT BRAIN 总被引:1,自引:0,他引:1
(1) The inhibitor of γ-aminobutyrate transaminase (GABA-T), amino-oxyacetic acid (AOAA), drastically reduced the activity of GABA-T to 30 per cent of the control value, with a corresponding increase of brain GABA, but had no effect on the activity of glutamate decarboxylase (GAD). (2) The monoamine oxidase (MAO) inhibitors phenelzine, phenylpropylhydrazine and phenylvalerylhydrazine, lowered GABA-T activity to 58, 49 and 48 per cent, respectively; this was associated with a marked elevation of brain GABA. (3) The action of phenelzine and phenylpropylhydrazine in vivo and in vitro could be abolished by pre-treatment of the tissue with the structurally related MAO inhibitors phenylisopropylhydrazine and trans-2-phenylcyclopropylamine. These had no action on the GABA system in vivo, either on the GABA content or on the GABA-T activity. These latter drugs, however, were unable to influence the effects of AOAA either on GABA or on GABA-T. (4) The possible mechanism of action on GABA and the enzyme activities of the GABA system is discussed. 相似文献
18.
Using [3H]naloxone at a concentration of 4.5 nm , the potent opiate agonist etorphine as well as the potent antagonist diprenorphine displace only about 75% of specific naloxone binding P2 fractions from rat whole forebrain, without additive effect. Several other opiates and antagonists completely displace specific naloxone binding. This indicates that etorphine and diprenorphine specifically bind to one and the same naloxone binding site (type I) while leaving another naloxone binding site (type II) unaffected. Type I binding sites are much more thermo-labile than type II. [3H]Naloxone binding to type I sites is unaffected by incubation temperature in the range 10 to 25°C. while binding type II sites decreases rapidly with increasing incubation temperature, no specific type II binding being detectable at or above 20°C. The two naloxone receptor types also differ with respect to pH dependence, and affinity for naloxone with types I and II having affinity constants (Kd) of 2 and 16 nm , respectively, at 0°C. The two binding sites have different regional distributions with high relative levels of type II receptors in cerebellum and low relative levels in pons-medulla and striatum. In whole rat brain there are about 4 times as many type II receptors as type I. These results suggest that naloxone and several other opiate agonists and antagonists bind to two distinct receptor types which are probably not agonist/antagonist aspects of the same receptor. 相似文献
19.
SOME EFFECTS OF DIETARY VITAMIN B6 DEFICIENCY AND 4-DEOXYPYRIDOXINE ON γ-AMINOBUTYRIC ACID METABOLISM IN RAT BRAIN 总被引:1,自引:1,他引:1
Abstract— Investigations of the respective effects of dietary vitamin B6 deficiency and 4-deoxypyridoxine (a vitamin B6 antagonist) on GABA metabolism in rat brain have been carried out. No convulsions were observed in rats subjected to either treatment. GABA levels were lowered by both treatments, the greatest diminutions being found with the dietary deficiency. Glutamic acid decarboxylase activity was reduced under both conditions, but the loss of activity in the B6 deficiency experiments could be attributed to cofactor depletion, whereas in the deoxypyridoxine experiments the loss of activity appears to be due to lower levels of available apoenzyme. The activity of GABA-transaminase was not affected by deoxypyridoxine treatment and only moderately reduced in the B.5 deficient animals. 相似文献
20.
The promethazine-sensitive binding of [3H]mepyramine to a membrane fraction from guinea-pig whole brain is saturable with a dissociation constant of 1.7 × 10-9M. The maximum amount of [3H]mepyramine binding varied widely between preparations, range 122–365 pmol/g protein, with a mean value of 227 ± 52 pmol/g protein. The inhibition of [3H]mepyramine binding by a number of drugs correlated closely with their potency as histamine H1 antagonists. (+) Chlorpheniramine was 240-fold more potent as an inhibitor of [3H]mepyramine binding than (-)-chlorpheniramine. All antagonists inhibited the binding of [3H]mepyramine to the same extent, but the Hill coefficients characterising the inhibition curves did not all approximate to unity, the value expected for a simple antagonist-receptor equilibrium. The distribution of histamine H1 receptors, defined by the promethazine-sensitive binding of [3H]mepyramine, in 11 different brain regions was uneven with the largest amounts in cerebellum, superior and inferior colliculus and hypothalamus and the smallest in caudate nucleus, brain stem and spinal cord. 相似文献