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Query: UNIPROT:P06889 (Mol)
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The effects of permeant cAMP analogs were studied on the function of the gamma-aminobutyric acidA (GABAA) receptor and on the activation of protein kinase A in brain synaptoneurosomes. Incubation of cerebral cortical synaptoneurosomes with permeant cAMP analogs decreased muscimol-induced 36Cl- uptake in a concentration-dependent manner. The order of potency was chlorophenylthio-cAMP (CPT-cAMP) greater than dibutyryl-cAMP greater than 8-bromo-cAMP. This order of potency was reflected by the ability of the analogs to gain access to the intravesicular compartment. cAMP, which failed to penetrate the membrane, had no effect. The half-maximal and maximal effects of the cAMP analogs were similar in the cerebral cortex, hippocampus, striatum, and cerebellum. To determine whether the cAMP analogs were acting through the activation of protein kinase A, protein kinase A activity was measured in lysed synaptoneurosomes, using kemptide as the substrate. In the lysed preparation, where the cAMP analogs have direct access to intracellular enzymes, the order of potencies of the cAMP analogs to activate protein kinase A (8-bromo-cAMP greater than CPT-cAMP greater than dibutyryl-cAMP) differed from the order of potencies to inhibit muscimol-induced 36Cl- uptake. In regional studies, the greatest effect of CPT-cAMP was observed in the cortex, whereas the smallest effect was observed in the hippocampus and cerebellum. To determine whether cAMP inhibition of GABA-gated ion flux was due to activation of protein kinase A, the time course for each response was measured. Inhibition of muscimol-induced 36Cl- uptake by cAMP analogs was nearly complete by 5 sec. Significant activation of protein kinase A by CPT-cAMP was also observed as early as 5 sec, but protein kinase A activation continued up to 10 min. The protein kinase inhibitor peptide inhibited protein kinase A activity in lysed synaptoneurosomes but had no effect on ion flux in intact synaptoneurosomes, as expected. However, a permeant kinase inhibitor, H-8, also failed to inhibit the effect of cAMP analogs on the muscimol response, yet it inhibited protein kinase A activity. The failure of H-8 to inhibit cAMP analog effects on GABAA receptor function was most likely due to the presence of ATP inside the synaptoneurosomes, because H-8 inhibition of protein kinase A was reduced in the presence of ATP. These results indicate that cAMP and cAMP analogs must penetrate the intravesicular compartment to inhibit GABAA receptor function. Although cAMP analogs decrease GABA-gated ion flux under conditions in which they activate protein kinase A, a causal relationship remains to be established.
Mol Pharmacol 1991 Mar
PMID:cAMP analogs inhibit gamma-aminobutyric acid-gated chloride flux and activate protein kinase A in brain synaptoneurosomes. 184 58

We have explored the functional significance of various drug-induced changes in t-[35S]butylbycyclophosporothionate (TBPS) binding to gamma-aminobutyric acidA (GABAA) receptors by comparing them with the actions of the drugs on GABA-induced 36Cl- uptake in rat cerebrocortical membrane preparations. In the presence of micromolar concentrations of GABA, various benzodiazepine receptor agonists, 3 alpha-21-dihydroxy-5 alpha-pregnan-20-one, and pentobarbital inhibited [35S]TBPS binding, whereas ethyl-beta-carboline-3carboxylate (beta-CCE), an inverse agonist, stimulated it, in general agreement with earlier reports [Mol. Pharmacol. 23:326-336 (1983); Mol. Pharmacol. 30:218-225 (1986)]. The drug-induced changes in [35S]TBPS binding, after normalization with respect to the corresponding action of diazepam, were closely related to the relative ability of the drugs to affect 36Cl- uptake, with a correlation coefficient of 0.98 and a slope of 0.85. Upon abolishment of GABA action by the use of bicuculline, however, all the tested drugs stimulated [35S]TBPS binding to various degrees, and their relative changes displayed a lower correlation coefficient of 0.69, with a slope of 2. In particular, the effects of the anesthetic steroid and pentobarbital on [35S]TBPS binding were markedly altered by GABA, which at 2 microM increased not only their maximal effects, but also their half-maximal concentrations severalfold. On the other hand, GABA did not significantly affect these parameters for diazepam under our experimental conditions. Also, the GABA-independent changes in [35S]TBPS binding produced by various benzodiazepine receptor agonists matched reasonably well the actions of the drugs on 36Cl- uptake, with a correlation coefficient of 0.85 and a slope of 1.0. These data suggest more pronounced functional coupling of the GABA sites with those for the steroid and the barbiturate, as compared with the benzodiazepine site. It appears that the degree of [35S]TBPS binding in the presence of GABA closely reflects the functional state of GABAA receptors and may be useful for characterization of allosteric interactions between various sites on the receptors.
Mol Pharmacol 1991 Mar
PMID:Correlation between gamma-aminobutyric acidA receptor ligand-induced changes in t-butylbicyclophosphoro[35S]thionate binding and 36Cl- uptake in rat cerebrocortical membranes. 184 61

Rats treated chronically with diazepam develop tolerance to diazepam effects and show changes in sensitivity of GABAergic systems. In order to investigate possible molecular mechanisms associated with these changes, we have evaluated the effects of acute and chronic diazepam treatment on levels of mRNA for the alpha 1 and beta 1 subunits of the GABAA receptor. Northern blots were hybridized with 32P-labeled GABA alpha 1 and beta 1 cDNA probes, and resulting bands were quantified by autoradiography and densitometry. Levels of alpha 1 mRNA were significantly decreased in cerebral cortex but not in cerebellum or hippocampus of chronic diazepam-treated rats. Acute diazepam treatment did not change levels of alpha 1 mRNA in any of the brain regions. Levels of beta 1 mRNA were examined by Northern blot analysis and also by solution hybridization analysis using a 32P-labeled riboprobe. Both methods showed that beta 1 mRNA was not significantly changed by chronic diazepam treatment. These results demonstrate a specific change in alpha 1 subunit that is associated with a state of altered GABA sensitivity and provide further support for the regional heterogeneity of chronic diazepam effects.
J Mol Neurosci 1990
PMID:Effects of continuous diazepam administration on GABAA subunit mRNA in rat brain. 196 63

1. The effects of baclofen and GABA on rat piriform cortex neurons were investigated electrophysiologically using a brain slice preparation. 2. At resting potential GABA depolarized and baclofen hyperpolarized the cell, probably through activation of Cl and K conductances acting at GABAA and GABAB receptors, respectively. 3. The GABAA receptors were concentrated on the apical and basal dendrites near the cell body, while the baclofen-sensitive GABA receptors were concentrated particularly on the basal dendrites. 4. The different distributions of receptor localization must have functional consequences which remain to be elucidated.
Cell Mol Neurobiol 1990 Dec
PMID:Differential effects of baclofen and gamma-aminobutyric acid (GABA) on rat piriform cortex pyramidal neurons in vitro. 196 26

In mammalian brain, the activation of GABAA-receptors is associated with the opening of chloride channels, whose function can be allosterically modulated by drugs, in particular by ligands of the benzodiazepine receptor. Agonistic ligands potentiate while inverse agonists reduce the efficiency of GABA. We have cloned cDNAs encoding alpha 1- and beta 1-subunits of the GABAA-receptor from rat brain. When the corresponding RNAs were co-expressed in Xenopus oocytes. GABA-induced currents were recorded which were inhibited by bicuculline and potentiated by pentobarbital. GABA activated the channel in a weakly cooperative manner. Furthermore, the GABA-response was modulated by benzodiazepine receptor ligands. However, not only various agonists but also the antagonist flumazenil and the inverse agonist DMCM potentiated the GABA-response. Thus, alpha 1- and beta 1-subunits are sufficient to form GABAA-receptors which contain benzodiazepine binding sites, although in a functionally restricted form.
Brain Res Mol Brain Res 1990 Aug
PMID:GABAA-receptor expressed from rat brain alpha- and beta-subunit cDNAs displays potentiation by benzodiazepine receptor ligands. 197 69

Plasma membrane potential generated by Na+, K(+)-ATPase provides the driving force for high-affinity, Na(+)-dependent uptake of glutamate into the cytoplasm of glutamatergic nerve endings and glial cells. Ca2(+)-calmodulin-dependent ATPase in the plasma membrane and Ca2(+)-ATPase in the endoplasmic reticulum influence the intracellular [Ca2+] and, therefore, the exocytotic release of neurotransmitter glutamate. The membrane potential across the membrane of the synaptic vesicles, generated by a H(+)-ATPase, provides the driving force for synaptic vesicular uptake of glutamate as well as that of GABA and glycine. Hypoxia and ischemia lead to release of glutamate, perhaps in consequence of an increased endogenous pool of glutamate and/or lack of substrate (ATP) for the ATPases. This release, rather than being exocytotic, is believed to result mainly from a reversal of the Na(+)-dependent high-affinity glutamate transporter in the plasma membrane.
Mol Chem Neuropathol 1990 Jan
PMID:Interrelationship between glutamate and membrane-bound ATPases in nerve cells. 198 May 85

Functional interactions between steroidal anesthetics and gamma-aminobutyric acidA (GABAA) receptors have been examined with 36Cl- uptake measurements in rat cerebrocortical synaptoneurosomes. The primary effect of the steroids was to enhance the affinity of GABA for its receptors without much effect on the maximal uptake rate; the ED50 for GABA decreased from 66.4 +/- 5.7 to 8.9 +/- 1.2 microM in the presence of 20 microM 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one. Stimulation of 36Cl- uptake by high concentrations of the anesthetic steroid in the absence of exogenous GABA was not due to direct stimulation of GABAA receptors, as currently proposed, but is due to enhanced action of endogenous GABA, inasmuch as the steroid markedly increases GABA affinity for the receptors. Typically, endogenous GABA was maintained at near 1 microM by a Na(+)-dependent GABA transport system in the synaptoneurosomes. Elevation of its level with nipecotic acid, a specific inhibitor of the GABA transport system, or reduction with GABase, a GABA-scavenging system, increased or decreased, respectively, the steroid-induced bicuculline-sensitive 36Cl- uptake. At low concentrations of GABA (less than 2 microM), the stimulatory effect of 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one was markedly potentiated by pentobarbital but antagonized by 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one, a partial agonist of higher affinity. These observations, along with the structure-activity relationships of steroid analogs, strongly suggest the existence of a specific binding site for the steroids in GABAA receptors and led us to propose a minimal model in which two key common functional groups of anesthetic steroids, 3 alpha-OH- and 17 beta-polar substituents, interact with GABAA receptors (probably through hydrogen bondings) while their hydrophobic backbone remains in contact with the fatty acyl chains of membrane phospholipids.
Mol Pharmacol 1990 Mar
PMID:Studies on the mechanism of interactions between anesthetic steroids and gamma-aminobutyric acidA receptors. 215 55

The action of TBPS (tert-butylbicyclophosphorothionate) on spontaneous chloride channels recorded from porcine pars intermediate lobe cells in primary culture has been studied. This compound, which binds specifically to the gamma-aminobutyric acidA (GABAA) receptor complex, is known as a channel-gating (non-competitive) GABA antagonist. The present results show that TBPS reduces spontaneous chloride channel activity in a dose-dependent manner, with an IC50 equal to 55 nM, which is a value comparable to its affinity for the GABAA binding sites. Single-channel analysis revealed that TBPS affects neither the amplitude nor the open time of these spontaneous channels but prolongs the longer closed times, resulting in a dramatic decrease in opening probability.
Mol Pharmacol 1990 Apr
PMID:Electrophysiological study of tert-butylbicyclophosphorothionate-induced block of spontaneous chloride channels. 215 64

In situ hybridization histochemistry and RNA blots were used to study expression of glutamic acid decarboxylase (GAD) mRNA in rat caudate-nucleus and substantia nigra. In situ hybridization combined with computerized image analysis revealed that in the intact substantia nigra reticulata the cross-section area of GAD mRNA positive neurons were 25% larger in the dorsolateral part as compared with the ventromedial part. A unilateral ibotenic acid injection in caudate-putamen lesioned neurons, some of which project to the ipsilateral substantia nigra. An increased level of GAD mRNA was observed in substantia nigra ipsilateral to the lesion. Computerized image analysis of sections from in situ hybridization revealed an increase in the number of silver grains over GAD mRNA positive neurons in the dorsolateral substantia nigra reticulata ipsilateral to the lesion. However, no change was observed in the ventromedial part suggesting that GAD mRNA expression in this part of the nigra is less sensitive to inhibition by caudate-putamen afferents. In agreement with in situ experiments, RNA blots showed a 2-fold increased level of GAD mRNA in substantia nigra ipsilateral to the lesion. The increased GAD mRNA expression in the deafferented substantia nigra suggests a disinhibition of nigral GABA neurons, resulting in an increased utilization of GABA in these substantia nigra neurons.
Brain Res Mol Brain Res 1990 Apr
PMID:Increased expression of glutamic acid decarboxylase mRNA in rat substantia nigra after an ibotenic acid lesion in the caudate-putamen. 215 80

The localization of gamma-aminobutyric acid-A (GABAA) receptors (GABAA-R) in the lower brainstem of the rat was examined by means of in situ hybridization histochemistry using an oligonucleotide probe to the sequence of the alpha 1 subunit (GABAA-R alpha 1). Strongly labeled neurons were found in the cranial motor nuclei, the dorsal motor nucleus of the vagus, reticular formation (large neurons), lateral vestibular nucleus, dorsal nucleus of the lateral lemniscus, central nucleus of the inferior colliculus, intermediate and white layers of the superior colliculus, red nucleus and substantia nigra. In addition, moderately labeled cells were abundant in the nucleus of the solitary tract, medial and inferior vestibular nuclei, parabrachial area, dorsal and ventral tegmental nuclei of Gudden, central gray matter, ventral nucleus of the lateral lemniscus, and reticular formation (small neurons). This study has therefore revealed some of the target neurons of GABA-containing fibers in the lower brainstem.
Brain Res Mol Brain Res 1990 May
PMID:Localization of GABAA-receptor alpha 1 subunit mRNA-containing neurons in the lower brainstem of the rat. 216 8


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