5β-Pregnan-3β-ol-20-one, a specific antagonist at the neurosteroid site of the GABAA receptor-complex

doi:10.1016/0304-3940(92)90454-F

Neuroscience Letters

Volume 135, Issue 2, 3 February 1992, Pages 273-275

https://doi.org/10.1016/0304-3940(92)90454-F Get rights and content

Abstract

Studies were made on the potentiation of [³H]flunitrazepam binding to rat brain membranes by γ-aminobutyric acid (GABA), pentobarbitone and pregnanolone (5β-pregnan-3α-ol-20-one). Epipreganolone, the 3β isomer of pregnanolone, inhibited competitively the potentiation by pregnanolone with a K_i of 10.5 μM without affecting that of GABA. The potentiation by pentobarbitone was slightly enhanced. Epipregnanolone alone showed only slight potentiation of benzodiazepine binding. These findings demonstrate that epipregnanolone is a specific antagonist of the neurosteroid site of the GABA_A receptor and raise the possibility of a physiological role for 3β-hydroxysteroids in modulating this receptor.

References (14)

E.F. Kirkness et al.
The stimulatory effects of secobarbital and pregnanolone on the GABA_A receptor can be blocked selectively
Eur. J. Pharmacol.
(1988)
M.D. Majewska
Antagonist-type interaction of glucocorticoids with the GABA receptor-coupled chloride channel
Brain Res.
(1987)
M.D. Majewska et al.
The neurosteroid dehydroepiandrosterone sulphate is an allosteric antagonist of the GABA_A receptor
Brain Res.
(1990)
R.M. McKernan et al.
GABA_A receptor subtypes immunopurified from rat brain with α-subunit specific antibodies have unique pharmacological properties
Neuron
(1991)
R.M. Atkinson et al.
Action of some steroids on the central nervous system of the mouse. II. Pharmacology
J. Med. Chem.
(1965)
N.L. Harrison et al.
Structure-activity relationship for steroid interaction with the γ-aminobutyric acid_A receptor complex
J. Pharmacol. Exp. Ther.
(1987)
M. Holzbauer
Physiological aspects of steroids with anaesthetic properties
Med. Biol.
(1976)

There are more references available in the full text version of this article.

Cited by (56)

Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABA<inf>A</inf>) receptors
2020, Journal of Biological Chemistry
Citation Excerpt :
It remains to be determined whether one or more of these “orphan” PAMs bind to the intrasubunit sites near the extracellular end of the TMD recently identified by photolabeling in αM4 by steroids containing photoreactive groups at C-21 or C-6 and in βM3 by a steroid containing a C-3α photoreactive group (25). Although early studies suggested that 3β-OH steroids competitively antagonize steroid enhancement of GABAAR function (53–55), subsequent studies indicate that they noncompetitively inhibit GABA responses in the absence of enhancing steroids, acting in a manner more similar to the sulfated 3β-OH neurosteroids PS and DHEAS (12, 56). Although steroid PAMs generally enhance [3H]muscimol or [3H]flunitrazepam equilibrium binding (51, 57, 58), our results indicate that inhibitory 3β-OH steroids can modulate [3H]muscimol binding in 3 different ways. (
Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABA_A receptors (GABA_ARs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABA_AR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABA_ARs by photoaffinity labeling with [³H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([³H]21-pTFDBzox-AP), a potent GABA_AR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M4 (β3Pro-415, β3Leu-417, and β3Thr-418) and M3 (β3Arg-309) helices located at the base of a pocket in the β⁺–α⁻ subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABA_AR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABA_AR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABA_AR β⁺–α⁻ subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.
Mechanisms of neurosteroid interactions with GABA<inf>A</inf> receptors
2007, Pharmacology and Therapeutics
Neuroactive steroids have some of their most potent actions by augmenting the function of GABA_A receptors. Endogenous steroid actions on GABA_A receptors may underlie important effects on mood and behavior. Exogenous neuroactive steroids have potential as anesthetics, anticonvulsants, and neuroprotectants. We have taken multiple approaches to understand more completely the interaction of neuroactive steroids with GABA_A receptors. We have developed many novel steroid analogues in this effort. Recent work has resulted in synthesis of new enantiomer analogue pairs, novel ligands that probe various properties of the steroid pharmacophore, fluorescent neuroactive steroid analogues, and photoaffinity labels. Using these tools, combined with receptor binding and electrophysiological assays, we have begun to untangle the complexity of steroid actions at this important class of ligand-gated ion channel.
The identification and simultaneous quantification of neuroactive androstane steroids and their polar conjugates in the serum of adult men, using gas chromatography-mass spectrometry
2007, Steroids
Certain androstane steroids (AS) modulate ionotropic receptors, as do the pregnane steroids. Whereas women produce significant amounts of neuroactive progesterone metabolites, the steroid neuromodulators in men originate mainly from the 3-oxo-4-ene C₁₉-steroids, which are converted to their 3α- and 3β-hydroxy-5α/5β-reduced metabolites. The neuromodulating effects of AS prompted us to monitor circulating levels of the steroids to estimate metabolic pathways in the periphery that may influence brain concentrations of AS. Hence, the serum levels of 20 steroids and 16 steroid polar conjugates including 17-oxo- and 17β-hydroxy-derivatives of 5α/β-androstane-3α/β-hydroxy-androstane steroids were quantified in 15 men (16–62 years of age) using GC–MS. The conjugated AS for the most part reached micromolar concentrations, these being two or three orders of magnitude higher than those of the free steroids. The ratios of conjugates to free steroids were one to two orders of magnitude higher than the values for the corresponding pregnane steroids. This data suggested that conjugation may considerably restrain the transport of free AS from the periphery into the central nervous system.
Neuroactive steroid effects on cognitive functions with a focus on the serotonin and GABA systems
2006, Brain Research Reviews
This article will review neuroactive steroid effects on serotonin and GABA systems, along with the subsequent effects on cognitive functions. Neurosteroids (such as estrogen, progesterone, and allopregnanolone) are synthesized in the central and peripheral nervous system, in addition to other tissues. They are involved in the regulation of mood and memory, in premenstrual syndrome, and mood changes related to hormone replacement therapy, as well as postnatal and major depression, anxiety disorders, and Alzheimer's disease. Estrogen and progesterone have their respective hormone receptors, whereas allopregnanolone acts via the GABA_A receptor. The action of estrogen and progesterone can be direct genomic, indirect genomic, or non-genomic, also influencing several neurotransmitter systems, such as the serotonin and GABA systems. Estrogen alone, or in combination with antidepressant drugs affecting the serotonin system, has been related to improved mood and well being. In contrast, progesterone can have negative effects on mood and memory. Estrogen alone, or in combination with progesterone, affects the brain serotonin system differently in different parts of the brain, which can at least partly explain the opposite effects on mood of those hormones. Many of the progesterone effects in the brain are mediated by its metabolite allopregnanolone. Allopregnanolone, by changing GABA_A receptor expression or sensitivity, is involved in premenstrual mood changes; and it also induces cognitive deficits, such as spatial-learning impairment. We have shown that the 3beta-hydroxypregnane steroid UC1011 can inhibit allopregnanolone-induced learning impairment and chloride uptake potentiation in vitro and in vivo. It would be important to find a substance that antagonizes allopregnanolone-induced adverse effects.
Discriminative stimulus effects of 5.6 mg/kg pregnanolone in DBA/2J and C57BL/6J inbred mice
2006, Alcohol
Citation Excerpt :
Amongst the isomers of pregnanolone with low efficacy at GABAA-receptor positive modulation, epipregnanolone had a much lower potency than pregnanolone, and epiallopregnanolone did not show substitution when compared across a similar dose range (Fig. 4). Both epiallopregnanolone and epipregnanolone are 3β-reduced hydroxysteroids, which have low efficacy at the GABAA receptor (Lambert et al., 1995; Purdy et al., 1990), and both act as competitive antagonists of neurosteroid-enhanced [3H] flunitrazepam binding (Prince & Simmonds, 1992, 1993). Data obtained from substitution of the isomers of pregnanolone show pregnanolone's cue to exhibit stereospecificity with the 3α-pregnane steroids being the most potent at producing pregnanolone-appropriate responding compared to the 3β-pregnane steroids.
Neurosteroids represent a class of endogenous compounds that exert rapid, nongenomic effects through neurotransmitter receptor systems such as γ-aminobutyric acid_A (GABA_A). Two neurosteroids, allopregnanolone (3α-hydroxy-5α-pregnan-20-one) and pregnanolone (3α-hydroxy-5β-pregnan-20-one), possess anxiolytic and sedative properties and show substitution for ethanol, benzodiazepines, and barbiturates in drug discrimination assays. A previous study examining the discriminative stimulus effects of 10 mg/kg pregnanolone in DBA/2J and C57BL/6J mice showed pregnanolone's discriminative stimulus to be mediated primarily through GABA_A positive modulation. This study examined the discriminative stimulus effects of a lower training dose (5.6 mg/kg) of pregnanolone in DBA/2J and C57BL/5J mice. Twelve male DBA/2J mice and 12 male C57BL/6J mice were trained to discriminate 5.6 mg/kg pregnanolone. GABA_A-receptor positive modulators, neuroactive steroids, NMDA receptor antagonists, and 5-HT₃ receptor agonists were tested for pregnanolone substitution. In DBA/2J and C57BL/6J mice benzodiazepine, barbiturate, and GABAergic neuroactive steroids all substituted for pregnanolone. In the DBA/2J mice, NMDA receptor antagonists showed generalization to the discriminative stimulus cues of pregnanolone, an effect not seen in the C57BL/6J mice. 5-HT₃ receptor agonists and zolpidem failed to substitute for pregnanolone's discriminative stimulus in either strain. AlloTHDOC and midazolam were more potent in producing pregnanolone-like discriminative stimulus effects in DBA/2J mice. These results provide a comprehensive look at pregnanolone's discriminative stimulus effects in two commonly used strains of mice. The present data suggest GABA_A-receptor positive modulation as the predominant receptor mechanism mediating the discriminative stimulus effects of pregnanolone. NMDA receptor antagonism was suggested in the DBA/2J mice and may represent a heterogenous cue produced by the lower training dose of pregnanolone.
Altered profiles of serum neuroactive steroids in premenopausal women treated for alcohol addiction
2005, Steroids
Long-term alcohol consumption results in menstrual irregularities due to the inhibition of progesterone secretion. Some progesterone metabolites, including three pregnanolone isomers (PI), abate, while pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) increase, alcohol tolerance. The rationale of this study was to evaluate how the neuroactive steroids reflect the impaired progesterone formation in premenopausal women treated for alcohol addiction, and whether detoxification therapy could restore female reproductive functions and psychosomatic stability by reinstatement of the steroid biosynthesis. Accordingly, serum allopregnanolone (3α-hydroxy-5α-pregnan-20-one (P3α5α)), pregnanolone (P3α5β), isopregnanolone (P3β5α) and epipregnanolone (P3β5β), progesterone, PregS, pregnenolone, 17α-hydroxy-pregnenolone (Preg17), 17α-hydroxy-progesterone (Prog17), DHEA, DHEAS, cortisol and estradiol were measured in 20 women during the therapy (start, 3 days, 14 days, 1 month, 4 months), and in 17 controls, using GC-MS or RIA and evaluated by age-adjusted ANCOVA with status and phase of the menstrual cycle (PMC) as factors, and status–PMC interaction.
The patients exhibited depressed progesterone, Prog17, PI, and estradiol, a decreased progesterone/pregnenolone ratio, a decreased ratio of neuroinhibiting P3α5α to neuroactivating PregS, and an elevated PregS and PregS/pregnenolone ratio. The treatment mostly restored the indices. The reduction of neuroinhibiting pregnanolone isomers in the patients is primarily associated with the impairment in ovarian steroid biosynthesis. Nevertheless, changes in enzyme activities connected with the formation of PI and the influence of altered physiological requirements on the balance between endogenous neuroinhibiting and neuroactivating steroids are also likely. The reinstatement of serum estradiol, progesterone, and PI during the therapy demonstrates its favorable effect on both reproductive functions and the psychosomatic stability of the patients.

View all citing articles on Scopus

View full text

5β-Pregnan-3β-ol-20-one, a specific antagonist at the neurosteroid site of the GABAA receptor-complex

Abstract

Eur. J. Pharmacol.

Brain Res.

Brain Res.

Neuron

Action of some steroids on the central nervous system of the mouse. II. Pharmacology

J. Med. Chem.

Structure-activity relationship for steroid interaction with the γ-aminobutyric acidA receptor complex

J. Pharmacol. Exp. Ther.

Physiological aspects of steroids with anaesthetic properties

Med. Biol.

5β-Pregnan-3β-ol-20-one, a specific antagonist at the neurosteroid site of the GABA_A receptor-complex

Structure-activity relationship for steroid interaction with the γ-aminobutyric acid_A receptor complex