Inhibition in the mammalian brain. A new theory of GABA mechanism of action.
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Biomedical subjects
A Cupello
Publications and source records attributed to A Cupello.
gamma-Aminobutyric acid stimulates chloride permeability across microdissected Deiters' neuronal membrane.
Fluxes of 36Cl- across freshly prepared Deiters' neuronal membranes have been studied in a two-compartment microchamber simulating the extra- and the intracellular space. The rate of 36Cl- influx was enhanced by gamma-aminobutyric acid (GABA) (10(-4) M), the effect being reversed by picrotoxin (10(-4) M) and by bicuculline (10(-5) M). Diazepam (10(-8)-10(-7) M) did not potentiate the response to GABA and rather depressed it. However, a barbiturate site is most probably present in the GABA receptor complexes since pentobarbitone (10(-4) M) was able to stimulate 36Cl- permeability to the same extent as GABA itself.
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gamma-Aminobutyric acid (GABA) removal from the synaptic cleft: a postsynaptic event?
In the present commentary we discuss the adequacy of Na+ transport-coupled presynaptic gamma-aminobutyric acid (GABA) uptake systems for the removal of GABA from the synaptic cleft. This discussion is based on the accepted stoichiometry for GABA presynaptic internalization, GABAout + 3Na+out + K+in in equilibrium GABAin + 3Na+in + K+out, on the parameters reported in the literature for typical synaptosomal preparations, and on the assumption that GABA removal must be a quick event (less than or equal to 2 msec), as derived from electrophysiological studies. On these bases, we have developed a calculation in order to evaluate the time course of synaptic cleft GABA removal by presynaptic systems and ended up with an overall value (t approximately 0.3 sec) which does not fit with the data derived from electrophysiological recordings. Moreover, we calculated that if such systems had the function of removing GABA within 2 msec, as it should be, a large depolarization would be brought about in GABAergic boutons, resulting ultimately in further GABA release. These considerations together with biochemical and pharmacological experimental results seem to exclude that presynaptic uptake systems have the function of removing GABA from the synaptic cleft. Our experimental data on the ability of a GABA-acceptive postsynaptic membrane (Deiters' neuron membrane) to transport GABA indicate that this system may have the correct characteristics for removing the neurotransmitter. This refers to both the kinetics and the electrophysiological consequences of the phenomenon.
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Asymmetric diffusion into the postsynaptic neuron: an extremely efficient mechanism for removing excess GABA from synaptic clefts on the Deiters' neurone plasma membrane.
Microdissected Deiters' neuron plasma membranes have been used for studying the passage of GABA through the membrane both in the inward and outward direction. Working with 0.2 mM GABA in the compartment simulating the outside of the neurone and with 2.0 mM GABA in the one simulating the inside we found a net transport of GABA towards the inside. This mechanism does not require a Na+ ion gradient across the membrane. The nature of the transport process involved was studied by determining the rate of [3H]-GABA inward passage as a function of GABA concentration (1 nM - 800 microM) on the outward side of the membrane. The results have shown that until 50 microM a diffusion process (v = D1 X C, where D1 = 3.1 X 10(-11) 1/micron 2 X sec) is the sole mechanism involved. Above 50 microM a second diffusion process is activated v = D2 X (C - 50 X 10(-6), where D2 = 2.8 X 10(-11) 1/micron 2 X sec. Taking in account both inward and outward directed diffusion, one can calculate 16 microM as the equilibrium concentration of GABA on the outward side of the membrane. From a kinetic point of view, these diffusion processes are able to reduce GABA concentration in a synaptic cleft from 3 mM to 20 microM within 3 mu sec. These diffusion systems are discussed as extremely efficient in removing the excess of released GABA in the synaptic cleft.
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Calcium ions in the presence of exogenous phosphatidylserine interfere with GABA diffusion through the Deiters' neuron membrane.
Diffusion of GABA through the plasma membrane of GABA-acceptive neurons might be a mechanism of importance for the termination of its synaptic action. In the present investigation we studied the effects of phosphatidylserine (PS) (10(-4)-10(-3) M), Ca2+ 2 mM and PS + 2 mM Ca2+ on such a process. The method involved the use of single microdissected Deiters' membranes which were put between two small microchambers in order to study the passage of GABA across the membrane. The results show that whereas PS and Ca2+ by themselves have no effect on such a process, PS + 2 mM CaCl2 give a significant, although slight, inhibition. The hypothesis that Calcium ion + PS effect is due to a disturbance of the interaction between GABA and endogenous PS molecules of the membrane is discussed.
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Pattern of labeling of rabbit cerebral cortex soluble proteins 24 hours after a single electroconvulsive shock.
The labeling of proteins in the particulate subcellular fractions and in the cell sap was studied in the cerebral cortex of rabbits 24 hours after a single electroconvulsive shock (ECS). To this end the animals were injected with [3H]valine subarachnoidally and sacrificed 30 minutes later. The incorporation of the labeled amino acid into proteins was slightly increased in all subcellular fractions but the increase was significant only for soluble proteins. Referring to the type of soluble polypeptides found in the cortex at this post ECS phase, no modification was found in the SDS electrophoresis pattern of steady state proteins. The fractionation pattern of labeled soluble proteins showed in one third (3 out of 9) of the experiments a remarkable stimulation of the synthesis of two polypeptides (25,000 and 54,000 Dalton MW), which were not labeled in the controls.
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Preincubation of homogenates from rat brain thalamus and striatum affects the rate of [3H]GABA uptake.
The effect of preincubation of brain homogenates on the subsequent uptake of [3H]GABA was studied in rat thalamus and corpus striatum. The results show that in both brain regions preincubation causes a decrease in the initial rate of [3H]GABA influx, the phenomenon being most evident in the thalamus.
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Neurotransmitter uptake in various brain regions of chronically morphinized rats.
Rats were long-term morphine-intoxicated by a fluid-diet model ensuring an equal nutrient intake in morphinized and control rats. Uptake of neurotransmitters and D-ala2-met5-enkephalinamide (Enk) was studied in the particulate fractions (obtained at 10,000 g) from well defined brain regions of long-term intoxicated and morphine withdrawn rats. In control animals the accumulation of [3H]glutamate and [3H]gamma-aminobutyric acid (GABA) reached the highest tissue/medium (T/M) ratio values, 30-120, in the regions studied while monoamine T/M values were 2-10. No active uptake of [3H]Enk could be demonstrated. Striatum showed the most evident modifications in neurotransmitter uptake. In this region the equilibrium T/M ratio for [3H]glutamate and [3H]GABA was significantly lower in intoxicated rats versus controls. Moreover, the T/M ratio for [3H]5-hydroxytryptamine (5-HT) was lower, while that for [3H]dopamine (DA) was higher in abstinent rats in comparison with the controls. [3H]glutamate and [3H]GABA uptakes were also significantly lower, respectively, in frontal cortex, hippocampus and brain stem in intoxicated rats, while [3H]5-HT uptake was significantly lower in hypothalamus after morphine withdrawal. The possible involvement of the endogenous opioid system in the etiology of the alterations is discussed.
Administration, Oral↗
Binding experiment KD values and physiologically active GABA concentrations: an only apparent contradiction?
An apparent contradiction exists between GABAA receptor binding KD's (less than 10(-6) M) and GABA concentrations physiologically active in various systems (greater than 10(-6) M). Taking in account that synaptic cleft GABA action and removal should be rather quick (within 2 ms) we show that in principle there is no contradiction between low KD values (100-150 nM) and high physiologically active GABA concentrations (greater than 40 microM).
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Evaluation of the electrophysiological consequences of GABA removal from the synaptic cleft by Na+ ion transport-coupled neuronal uptake.
The pre- and postsynaptic electrophysiological consequences of a carrier-mediated, Na+ ion transport-coupled removal of gamma-aminobutyric acid (GABA) from the relevant synaptic clefts are discussed. Assuming for the GABA internalization process a stoichiometry like GABAo + 3NA+o + K+i in equilibrium GABAi + 3Na+i + K+o and a synaptic cleft GABA maximal concentration of 100 microM we calculated the presynaptic depolarization associated with GABA removal between 11.5 and 38.2 mV. At the postsynaptic level the effect appears to be less marked.
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A calculation method for evaluating the time course of GABA removal from a synaptic cleft by presynaptic uptake systems.
A calculation method for evaluating the time course of gamma-aminobutyric acid (GABA) removal from a synaptic cleft by presynaptic uptake is suggested. The evaluation of the actual time required to remove GABA requires the knowledge of: (a) KM's and Vmax's (mol/min/mg protein) of the synaptosomal uptake systems in a certain brain area; (b) the synaptosomal volume per mg of protein in the synaptosomal preparation used; (c) the mean sphere diameter for synaptic boutons in the brain area considered and the proportion of GABAergic nerve terminals.
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Increased binding of GABA to its post-synaptic carrier sites on the plasma membrane of Deiters' neurons after a learning experiment in rats.
A new micromethod for studying the interaction of gamma-aminobutyric acid (GABA) with its post-synaptic uptake in a defined type of nerve cell is described. The method involves the isolation by free hand microdissection of Deiters' nerve cells from the rabbit lateral vestibular nucleus and their incubation with tritiated GABA in the presence of 100 mM Na+ at 4 degrees C. From the binding data a Kd was calculated for this interaction of 104 nM and a BMax of 8.4 X 10(5) sites/neuron. The method was applied to the study of the modifications of the binding of GABA to Deiters' neurons from control rats and rats trained to balance on a steel wire in order to reach food. This performance is a powerful stimulation to the vestibular system. The results show that, in the binding experiment with 100 nM [3H]GABA, the amount of GABA which specifically binds to the Deiters' neurons is increased by 38% in the trained rats. Analogously, when the incubation with GABA was performed at 37 degrees C, involving an intake of GABA into the neurons, the amount of GABA taken up increases by 50% in the trained group. No GABA-binding or uptake increase was found when animals were subjected to intense vestibular stimulation for a short period without learning. These results indicate that when rats learn a behavioral test which involves an improved vestibular control, there is a specific neurochemical modification in the neurons of the lateral vestibular nucleus. This modification seems to be of importance for the physiology of the neuronal circuits controlling the vestibular function in the rat.
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Inhibition by sodium valproate of the transport of GABA through the Deiters' neurone plasma membrane.
The transport of GABA through the microdissected plasma membrane Deiters' neurone reflects the physiological event of postsynaptic uptake of GABA by its uptake carrier. Sodium valproate at concentrations greater than or equal to 2.4 mM was able to decrease markedly (57%) such a transport. This effect, which reduces the efficiency of the GABA postsynaptic inactivation process, might be a mechanism for the potentiation by valproate of the synaptic action of GABA.
Animals↗
Labeling of RNA in young and adult rat brain: evidence for different RNA processing.
The labeling of RNA in young and adult rat brain has been studied by measuring in vitro (tissue slices incubation) the incorporation of labeled uridine into RNA of total tissue and of the various subcellular fractions purified from cerebral hemispheres of 1- and 10-month-old rats. Gel electrophoretic analysis of the newly synthesized nuclear and microsomal RNA was also accomplished. An active metabolism of RNA in adult animals was found; moreover, distinct differences in ribosomal RNA processing in cerebral hemispheres of 1- and 10-month-old rats, with a more rapid processing in the brain of adult animals, were obtained.
Aging↗
Dose and time dependent effects of morphine on the incorporation of [3H]valine into soluble brain and liver proteins.
Morphine (10(-6)-10(-5) M) causes an increase in incorporation of [3H]valine into soluble proteins during 4 hr in rat brain cortical slices, liver slices and cultivated astroglial cells. The effects are dose-dependent. They are neither cell specific nor strictly related to classical opiate receptors. Pulse-labeling with [3H]valine for 60 min after incubation in 10(-6)-10(-5) M morphine, resolves time-dependent changes in incorporation, with both increases and decreases in protein metabolism.
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Labeling of poly(A) associated RNA in synaptosomes and the other subcellular fractions of rat cerebral cortex in basal conditions and during training.
The labeling of total and poly(A) associated RNA from the subcellular fractions of cerebral cortex was studied after a 1-hour 3H-uridine pulse in control and trained rats. In the control animals, the fraction of newly synthesized brain cortex RNA which is found in synaptosomes is the minimal one. However, in synaptosomes the proportion of poly(A)+ RNA within newly synthesized RNA is maximal. Training of the rats does not influence total incorporation of label into RNA in the brain cortex and the distribution of newly synthesized RNA within the subcellular fractions. The proportions of poly(A)+ RNA within total labeled RNA in the various fractions are also unaffected.
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Labeling of non poly(A) associated RNA in the rabbit cerebral cortex 24 hours after a single electroconvulsive shock.
The labeling pattern of non poly(A) associated (poly(A)-)RNA of rabbit cerebral cortex was studied 24 hr after a single electroconvulsive shock (ECS). The animals were injected subarachnoidally with [3H]uridine and sacrificed 1 hr later. The fractionation pattern of labeled nuclear poly(A)-RNA in the cerebral cortex of ECS treated animals was identical to that of the controls. However, microsomal poly(A)-RNA from the treated animals showed an increased labeling of 18S ribosomal RNA. Also 28S RNA displayed a higher labeling but the effect was not statistically significant. These results indicate a more efficient production of ribosomal RNA in the late post-ECS period which might be in relationship with an increased activity of brain protein synthesis machinery.
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Labeling of rabbit cerebral cortex poly(A) RNA 24 hours after a single electroconvulsive shock.
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