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GABA-site antagonism and pentobarbital actions do not depend on the α-subunit type in the recombinant rat GABA-A receptor
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
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2006 (English)In: Acta Physiologica, ISSN 1748-1708, E-ISSN 1748-1716, Vol. 187, no 4, 479-488 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2006. Vol. 187, no 4, 479-488 p.
Identifiers
URN: urn:nbn:se:umu:diva-2300DOI: 10.1111/j.1748-1716.2006.01593.xOAI: oai:DiVA.org:umu-2300DiVA: diva2:140237
Available from: 2007-05-04 Created: 2007-05-04 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Effects of neuroactive steroids on the recombinant GABAA receptor in Xenopus oocyte
Open this publication in new window or tab >>Effects of neuroactive steroids on the recombinant GABAA receptor in Xenopus oocyte
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Introduction: Neuroactive steroids represent a class of both synthetic and naturally occurring steroids that have an effect on neural function. In addition to classical genomic mechanism by the hormones progesterone, deoxycorticosterone and testosterone 3α-OH metabolites of these hormones enhance GABAA receptor through rapid non-genomic mechanism. The site(s) of action of these neuroactive steroids namely 3α-OH-5α-pregnan-20 one, (3α,5α)-3,21-deoxycorticosterone(3α5α-THDOC) and 5α androstane-3α,17β-diol on GABAA receptor are distinct from that of benzodiazepines and barbiturate binding sites. The modulation site(s) has a well-defined structure activity relationship with a 3α-hydroxy and a 20-ketone configuration in the pregnane molecule required for agonistic action. Pregnenolone sulfate is a noncompetitive GABAA receptor antagonist and inhibit GABA activated Cl- current in an activation dependant manner. 3β-hydroxy A-ring reduced pregnane steroids are also GABAA receptor antagonist and inhibit GABAA receptor function and its potentiation induced by their 3α-diesteromers in a noncompetitive manner.

Aim: The aim was to investigate if the effect of GABA, pentobarbital antagonism by bicuculline and if the effect of GABA-agonist and antagonist neuroactive steroids including pregnenolone sulfate is dependant on the α-subunits of GABAA receptor. Furthermore, the studies aimed at investigating the binding site of pregnenolone sulfate and if its effect is dependent on γ-subunit. In addition, the inhibitory effect of pregnenolone sulfate and 3β-hydroxy steroids has been characterized. We also wanted to investigate if the neuroactive steroids effect vary between the human and rat recombinant α1β2γ2L receptors and between the long (L) and short (S) variants of γ2-subunit.

Method: Experiments were performed by the two electrodes voltage-clamp technique using oocytes of Xenopus laevis expressed with recombinant GABAA receptors containing α1, α4 or α5, β2, γ2L and γ2S-subunits.

Results: There was no difference between the α1, α4 and α5-containing subunits regarding GABA and pentobarbital inhibition by bicuculline. GABA-activated current in the binary αβ was potent than that of ternary αβγ receptor. Unlike Zn2+ effect, inhibition by pregnenolone sulfate on the GABAA receptor is not dependant on the γ-subunit. It is likely that the 2’ residue closest to the N-terminus of the protein at M2 helix on both α1 and β2 subunit are critical to the inhibitory actions of PS and the function of Cl- channels. Point mutation at M2 helix of the β2-subunit (b2A252S) can dramatically reduce the inhibitory effect of PS on the GABAA receptors without affecting the inhibitory properties of 3β-hydroxysteroids. Agonist and antagonist steroids also varied in their efficacy between the human and rat α1β2γ2L receptor. Neuroactive steroids also showed difference between human γ2L and γ2S-containing receptor.

Conclusions: GABA and pentobarbital antagonism by bicuculline is not dependant on α-subunit. Pregnenolone sulfate binding site is different from that of Zn2+. 3β-hydroxysteroids and pregnenolone sulfate inhibit GABAA receptor through different mechanisms. Neuroactive steroids also differ between species and between the long and short variant of γ- subunit.

Place, publisher, year, edition, pages
Umeå: Medicinsk fakultet, 2007. 72 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1099
Keyword
GABA, GABAA receptor, neuroactive steroids
National Category
Clinical Science
Identifiers
urn:nbn:se:umu:diva-1112 (URN)978-91-7264-273-7 (ISBN)
Public defence
2007-05-25, D, by 1D, Tr 9 (tandläkerhögskolan), NUS, 13:00 (English)
Opponent
Supervisors
Available from: 2007-05-04 Created: 2007-05-04 Last updated: 2016-06-15Bibliographically approved
2. Progesterone metabolites: learning, tolerance, antagonism & metabolism
Open this publication in new window or tab >>Progesterone metabolites: learning, tolerance, antagonism & metabolism
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Progesterone metabolites as allopregnanolone, isoallopregnanolone and tetrahydrodeoxy-corticosterone (THDOC) are increased in the luteal phase of the menstrual cycle, throughout pregnancy and during stress. Allopregnanolone and THDOC are neurosteroids with 3α-hydroxy, 5α-configurations and positive modulating effect on the GABAA receptor. They have similar properties and effect, and share the same binding sites on the GABAA receptor. Isoallopregnanolone has a 3β-hydroxy, 5α-configuration and a diverse effect as a proposed antagonist to both allopregnanolone and THDOC. Neurosteroids are thought to exert their effect predominantly at extrasynaptic GABAA receptors, containing for example α4- or α5-subunits. Such receptors are involved in the tonic response. Different subunits have diverse distribution pattern in the brain and are involved in different functions. The α5-subunit, mainly expressed in the hippocampus, is involved in learning, while α4 is more widespread and involved in e.g. anxiety and anaesthesia.

The aim of the present thesis was to contribute to the knowledge about selected progesterone metabolites and their effects on learning and tolerance development, as well as their metabolism. Also basic characteristics between different α-subunits of the GABAA receptor were evaluated.

The thesis shows that the effect of bicuculline and pentobarbital is not dependent on the α-subunit isoform of the GABAA receptor expressed in oocytes. Acute tolerance developed after allopregnanolone-induced anaesthesia with a decrease at both mRNA and protein levels of the GABAA receptor α4-subunit in the thalamus VPM nucleus. A negative correlation between the α4 mRNA and the increased dose of allopregnanolone needed to maintain the anaesthesia level was also shown. In addition, allopregnanolone induces a learning impairment in the Morris water maze test, when high concentrations of allopregnanolone are present in the brain. This impairment is not possible to reverse by isoallopregnanolone. In α5β3γ2L-transfected HEK-293 cells THDOC induces a baseline shift of its own and also potentiate the GABA-current. Neither of those THDOC effects can be inhibited by isoallopregnanolone. Instead isoallopregnanolone shows an agonistic effect on the THDOC-potentiation of the GABA-response. The main allopregnanolone metabolites identified, 5α-DHP and isoallopregnanolone, as well as allopregnanolone itself are mainly localized to the brain after an i.v. injection. After an isoallopregnanolone injection there is a more even distribution of the given steroid and the metabolites between plasma and brain. There is an epimerisation between isoallopregnanolone and allopregnanolone and vice versa.

In conclusion, the present thesis shows that the α4-subunit in the thalamus VPM nucleus is likely to be involved in the acute tolerance development against allopregnanolone and that allopregnanolone-induced learning impairment is likely to be hippocampus dependent. The lack of antagonistic effect of isoallopregnanolone on the THDOC-induced α5β3γ2L-GABAA response, together with epimerisation of isoallopregnanolone to allopregnanolone, could explain why isoallopregnanolone does not work as an antagonist to the allopregnanolone-induced learning impairment in a hippocampus dependent learning task.

Place, publisher, year, edition, pages
Umeå: Umeå university, 2009. 73 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1306
Keyword
allopregnanolone, isoallopregnanolone, THDOC, learning, tolerance, antagonism, inhibition, patch clamp, Morris water maze, metabolism, epimerization
National Category
Obstetrics, Gynecology and Reproductive Medicine
Research subject
Obstetrics and Gynaecology
Identifiers
urn:nbn:se:umu:diva-27064 (URN)978-91-7264-888-3 (ISBN)
Public defence
2009-12-05, Sal 914, By 1, 9 tr, NUS, Norrlands Universitetssjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2009-11-16 Created: 2009-11-09 Last updated: 2010-01-18Bibliographically approved

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Mozibur, RahmanLindblad, CharlotteJohansson, Inga-MajHolmberg, ElinorBäckström, TorbjörnWang, Ming-De
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