Identification of α2‐ and α3‐subunits of the GABAA‐benzodiazepine receptor complex purified from the brains of young rats

Fuchs, Karoline; Adamiker, D; Sieghart, Werner

doi:10.1016/0014-5793(90)80634-u

Cited by 46 publications

(21 citation statements)

References 15 publications

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“…The ␣ 3 subunit is also contained in a significant proportion of GABA A receptors (22,24,25). To investigate whether 3␤-HSD-containing neurons also express GABA A receptors, we have used a specific polyclonal antibody against the ␣ 3 subunit (17,18,26) and a monoclonal antibody against the ␤ 2 ͞␤ 3 subunits that also cross-reacts with the ␣ 1 subunit (24,27,28). In a recent report, this latter antibody has been used successfully to localize the GABA A receptor complex in the brain of the frog Rana pipiens (29).…”

Section: Discussionmentioning

confidence: 99%

γ-Aminobutyric acid, acting through γ-aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus

do-Rego

Mensah-Nyagan

Beaujean

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Most of the actions of neurosteroids on the central nervous system are mediated through allosteric modulation of the ␥-aminobutyric acid type A (GABA A) receptor, but a direct effect of GABA on the regulation of neurosteroid biosynthesis has never been investigated. In the present report, we have attempted to determine whether 3␤-hydroxysteroid dehydrogenase (3␤-HSD)-containing neurons, which secrete neurosteroids in the frog hypothalamus, also express the GABAA receptor, and we have investigated the effect of GABA on neurosteroid biosynthesis by frog hypothalamic explants. Double immunohistochemical labeling revealed that most 3␤-HSD-positive neurons also contain GABAA receptor ␣3 and ␤2͞␤3 subunit-like immunoreactivities. Pulse-chase experiments showed that GABA inhibited in a dose-dependent manner the conversion of tritiated pregnenolone into radioactive steroids, including 17-hydroxy-pregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, and dihydrotestosterone. The effect of GABA on neurosteroid biosynthesis was mimicked by the GABA A receptor agonist muscimol but was not affected by the GABA B receptor agonist baclofen. The selective GABAA receptor antagonists bicuculline and SR95531 reversed the inhibitory effect of GABA on neurosteroid formation. The present results indicate that steroid-producing neurons of the frog hypothalamus express the GABAA receptor ␣3 and ␤2͞␤3 subunits. Our data also demonstrate that GABA, acting on GABA A receptors at the hypothalamic level, inhibits the activity of several key steroidogenic enzymes, including 3␤-HSD and cytochrome P450C17 (17␣-hydroxylase).neuroactive steroids ͉ 3␤-hydroxysteroid dehydrogenase ͉ GABA A-benzodiazepine receptor complex

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Section: Discussionmentioning

confidence: 99%

γ-Aminobutyric acid, acting through γ-aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus

do-Rego

Mensah-Nyagan

Beaujean

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…8,16 The a1 and a2 antibodies were coupled to keyhole limpet hemocyanine. The a1 antibody was directed against the N-terminus and contained a C-terminal cysteine.…”

Section: Polyclonal Gaba a Receptor Subunit Antibodiesmentioning

confidence: 99%

“…The specificity of each GABA A receptor subunit antibody was reported previously. 16,41,61,70,79 The monoclonal anti-actin antibody used for western blot analysis, as a control for total protein, binds to a highly conserved region of actin, an abundant cytoskeletal protein.…”

Section: Polyclonal Gaba a Receptor Subunit Antibodiesmentioning

confidence: 99%

Benzodiazepine-mediated regulation of α1, α2, β1–3 and γ2 GABAA receptor subunit proteins in the rat brain hippocampus and cortex

et al. 1999

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Abstract-Prolonged flurazepam exposure regulates the expression of selected (a1, b2, b3) GABA A receptor subunit messenger RNAs in specific regions of the hippocampus and cortex with a time-course consistent with benzodiazepine tolerance both in vivo and in vitro. In this report, the immunostaining density of six specific GABA A receptor subunit (a1, a2, b1-3 and g2) antibodies was measured in the hippocampus and cortex, among other brain areas, in slide-mounted brain sections from flurazepam-treated and control rats using quantitative computer-assisted image analysis techniques. In parallel with the localized reduction in a1 and b3 subunit messenger RNA expression detected in a previous study, relative a1 and b3 subunit antibody immunostaining density was significantly decreased in flurazepam-treated rat hippocampal CA1, CA3 and dentate dendritic regions, and in specific cortical layers. Quantitative western blot analysis showed that b3 subunit protein levels in crude homogenates of the hippocampal dentate region from flurazepam-treated rats, an area which showed fairly uniform decreases in b3 subunit immunostaining (16-21%), were reduced to a similar degree (18%). The latter findings provide independent support that relative immunostaining density may provide an accurate estimate of protein levels. Consistent with the absence of the regulation of their respective messenger RNAs immediately after ending flurazepam administration, no changes in the density of a2, b1 or b2 subunit antibody immunostaining were found in any brain region. g2 subunit antibody staining was changed only in the dentate molecular layer.The selective changes in GABA A receptor subunit antibody immunostaining density in the hippocampus suggested that a change in the composition of GABA A receptors involving specific subunits (a1 and b3) may be one mechanism underlying benzodiazepine anticonvulsant tolerance. ᭧ 1999 IBRO. Published by Elsevier Science Ltd.Key words: tolerance, flurazepam, quantitative immunohistochemistry, dentate gyrus, limbic system, CA1 pyramidal cells.Although effective anxiolytics and hypnotics, the clinical use of benzodiazepines as anticonvulsants is limited by tolerance development during prolonged administration. A large body of evidence has indicated that benzodiazepine tolerance is related to a reduction in GABA A receptor-mediated fast inhibitory synaptic transmission (for reviews see Refs 2 and 27). The native GABA A receptor is a pentamer composed of combinations of homologous proteins derived from five subunit families with multiple variants (a1-6, b1-4, g1-3, d1 and e1). 8,11,34 The GABA A receptor macromolecule contains binding domains for GABA and several allosteric modulators, including a well-characterized benzodiazepine binding site. 59Benzodiazepines potentiate GABA A receptor-mediated inhibitory function by increasing the opening probability of the integral Cl Ϫ channel, 52,62 and may increase Cl Ϫ channel conductance. 13Although the synaptic mechanisms mediating benzodiazepine tolerance have not been...

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“…13, 14; see also Benke et al, 1991;Gambarana et al, 1991;. These expression changes are therefore consistent with (1) type II and type I BZ receptors predominating in neonatal and adult cortex, respectively (Lippa et al, 1981;Chisholm et al, 1983); (2) zolpidem having very low affinity for P6 rat brain homogenates (Sieghart and Schlerka,199 1); and (3) 3H-flunitrazepam photolabeling mainly three (Y subunit proteins (55, 59, 62 kDa) in neonatal cortex, and mainly one (51 kDa) in adult cortex (Eichinger and Sieghart, 1986;Sato and Neale, 1989;Fuchs et al, 1990;Vitorica et al, 1990). …”

Section: Cortical Receptorsmentioning

confidence: 99%

The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development

1992

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The embryonic and postnatal expression of 13 GABAA receptor subunit genes in the rat CNS was studied by in situ hybridization. Each transcript exhibited a unique regional and temporal developmental expression profile. For example, in both embryonic and early postnatal cortex and thalamus, expression of the alpha 2, alpha 3, alpha 5, and beta 3 mRNAs was pronounced. In particular, the alpha 5 gene expression underwent a prominent peak in early brain. Subsequently, the thalamocortical expression of these four genes substantially diminished and was superseded in the adult by the alpha 1, alpha 4, beta 2, and delta subunit mRNAs. Similarly, gamma 1 and gamma 3 gene expression also dropped markedly during development, their initial stronger expression being restricted to relatively few structures. In contrast, gamma 2 gene expression was widespread and mostly remained constant with increasing age. The medial septum and globus pallidus were regions expressing few subunits in both early postnatal and adult stages, allowing clear developmental combinatorial changes to be inferred (alpha 2/alpha 3 beta 2 gamma 2 to alpha 1 beta 2 gamma 2, alpha 2/alpha 3 beta 2 gamma 1 to alpha 1 beta 2 gamma 1/gamma 2, respectively). In contrast, cerebellar Purkinje cells exhibited no developmental switch, expressing only the alpha 1, beta 2, beta 3, and gamma 2 mRNAs from birth to adult. Certain GABAA transcripts were also detected in germinal zones (e.g., beta 1, beta 3, gamma 1) and in embryonic peripheral tissues such as dorsal root ganglia (e.g., alpha 2, alpha 3, beta 3, gamma 2) and intestine (gamma 3). Some parallels in regional and temporal CNS expression were noted (e.g., alpha 1 beta 2, alpha 2 beta 3, alpha 4/alpha 6 delta), whereas the alpha 5 and beta 1 regional mRNA expressions converted over time. The changes of GABAA receptor subunit gene expression suggest a molecular explanation for earlier observations on changing ligand binding affinities. Thus, the composition, and presumably properties, of embryonic/early postnatal rat GABAA receptors differs markedly from those expressed in the adult brain.

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Identification of α₂‐ and α₃‐subunits of the GABA_A‐benzodiazepine receptor complex purified from the brains of young rats

Cited by 46 publications

References 15 publications

γ-Aminobutyric acid, acting through γ-aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus

γ-Aminobutyric acid, acting through γ-aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus

Benzodiazepine-mediated regulation of α1, α2, β1–3 and γ2 GABAA receptor subunit proteins in the rat brain hippocampus and cortex

The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development

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