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, 10 (3), e0120473
eCollection

The Non-Benzodiazepine Anxiolytic Drug Etifoxine Causes a Rapid, Receptor-Independent Stimulation of Neurosteroid Biosynthesis

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The Non-Benzodiazepine Anxiolytic Drug Etifoxine Causes a Rapid, Receptor-Independent Stimulation of Neurosteroid Biosynthesis

Jean Luc do Rego et al. PLoS One.

Abstract

Neurosteroids can modulate the activity of the GABAA receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABAA receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism.

Conflict of interest statement

Competing Interests: The authors have no conflict of interest to declare. Jean Luc do Rego, David Vaudry and Hubert Vaudry have not received any financial compensation or salary support for this study.

Figures

Fig 1
Fig 1. Simplified diagram recapitulating the biosynthetic pathways of neurosteroids in the brain of vertebrates.
HST, hydroxysteroid sulfotransferase; P450AROM, cytochrome P450 aromatase; P450scc, cytochrome P450 side-chain cleavage; P450C17, cytochrome P450 17α-hydroxylase / C17,20-lyase; STS, sulfatase; 3α-HSD, 3α-hydroxysteroid dehydrogenase; 3β-HSD, 3β-hydroxysteroid dehydrogenase; 5α-R, 5α-reductase; 17β-HSD, 17β-hydroxysteroid dehydrogenase.
Fig 2
Fig 2. Analysis of radioactive steroids formed after a 2-h incubation of frog hypothalamic explants with tritiated pregnenolone ([3H]Δ5P) in the absence (A) or presence of 3x10-6 M etifoxine (B).
The ordinate indicates the radioactivity measured in the HPLC eluent. The dashed lines represent the gradient of secondary solvent (% solution B). The arrows indicate the elution positions of standard steroids: 17OH-Δ 5P, 17-hydroxypregnenolone; DHEA, dehydroepiandrosterone; Δ 4, androstenedione; 17OH-P, 17-hydroxyprogesterone; tetrahydrodeoxycorticosterone, THDOC; P, progesterone; Δ 5P, pregnenolone; DHP, dihydroprogesterone; THP, tetrahydroprogesterone.
Fig 3
Fig 3. Effect of graded concentrations of etifoxine on the conversion of tritiated pregnenolone ([3H]Δ5P) into 17-hydroxypregnenolone (17OH-Δ5P), dehydroepiandrosterone (DHEA), progesterone (P), dihydroprogesterone (DHP) and tetrahydroprogesterone (THP) by frog hypothalamic explants (duration of the incubation: 2h).
The values were calculated from the areas under the peaks in chromatograms similar to those presented in Fig. 1. Results are expressed as percentages of the amount of each steroid formed in the absence of etifoxine. Values are the mean (± SEM) of four independent experiments. *p<0.05; **p<0.01; ***p<0.001; ns, not statistically different from control (C).
Fig 4
Fig 4. Time-course of the conversion of tritiated pregnenolone ([3H]Δ5P) into radioactive 17-hydroxypregnenolone (17OH-Δ5P), dehydroepiandrosterone (DHEA), progesterone (P), dihydroprogesterone (DHP) and tetrahydroprogesterone (THP) by frog hypothalamic explants in the absence (○) or presence of 3x10-6 M etifoxine (●).
The values were calculated from the areas under the peaks in chromatograms similar to those presented in Fig. 1. Results are expressed as percentages of the amount of each steroid formed compared to the total amount of radiolabeled compounds resolved by HPLC analysis including [3H]Δ 5P. Values are the mean (± SEM) of four independent experiments. *p<0.05; **p<0.01; ***p<0.001 compared to respective control values; ns, not statistically different (one-way ANOVA followed by a post hoc Dunnett’s test).
Fig 5
Fig 5. Effects of etifoxine (3x10-6 M) in the absence or presence of the TSPO antagonist PK11195 (3x10-5 M), the central-type benzodiazepine receptor antagonist flumazenil (3x10-5 M) or the GABAA receptor antagonist bicuculline (3x10-5 M) on the conversion of tritiated pregnenolone ([3H]Δ5P) into 17-hydroxypregnenolone (17OH-Δ5P), dehydroepiandrosterone (DHEA), progesterone (P) and tetrahydroprogesterone (THP) by frog hypothalamic explants.
The values were obtained from experiments similar to those presented in Fig. 1. Results are expressed as percentages of the amount of each steroid formed in the absence of drugs. Values are the mean (± SEM) of four independent experiments. *p<0.05; **p<0.01; ***p<0.001 compared to respective control values; NS, not statistically different from control; ns, not statistically different from etifoxine-stimulated level (one-way ANOVA followed by a post hoc Student-Newman-Keul’s test).
Fig 6
Fig 6. Effects of etifoxine (10-6 M) in the absence or presence of triakontatetraneuropeptide (TTN), a specific TSPO agonist (3x10-8 M), on the conversion of tritiated pregnenolone ([3H]Δ5P) into 17-hydroxypregnenolone (17OH-Δ5P), dehydroepiandrosterone (DHEA), progesterone (P) and tetrahydroprogesterone (THP) by frog hypothalamic explants.
The values were obtained from experiments similar to those presented in Fig. 1. Results are expressed as percentages of the amount of each steroid formed in the absence of drugs. Each value is the mean (± SEM) of four independent experiments. **p<0.01, ***p<0.001 vs control; ##p<0.01, ###p<0.001 vs etifoxine alone; §§ p<0.01, §§§ p<0.001 vs TTN alone (one-way ANOVA followed by a post hoc Student-Newman-Keul’s test).
Fig 7
Fig 7. HPLC analysis of radioactive steroids formed after a 1-h incubation of frog hypothalamic homogenates with tritiated pregnenolone ([3H]Δ5P) in the absence (A) or presence of 10-6 M etifoxine (B).
The ordinate indicates the radioactivity measured in the HPLC eluent. The dashed lines represent the gradient of secondary solvent (% solution B). The arrows indicate the elution positions of standard steroids: 17OH-Δ 5P, 17-hydroxypregnenolone; DHEA, dehydroepiandrosterone; Δ 4, androstenedione; 17OH-P, 17-hydroxyprogesterone; tetrahydrodeoxycorticosterone, THDOC; P, progesterone; Δ 5P, pregnenolone; DHP, dihydroprogesterone; THP, tetrahydroprogesterone.
Fig 8
Fig 8. Effects of etifoxine (10-6 M and 3x10-6 M) or triakontatetraneuropeptide (TTN, 10-6 M) on the conversion of tritiated pregnenolone into 17-hydroxypregnenolone (17OH-Δ5P), dehydroepiandrosterone (DHEA), progesterone (P), dihydroprogesterone (DHP) and tetrahydroprogesterone (THP) by frog hypothalamic homogenates (duration of the incubation: 1h).
The values were calculated from the areas under the peaks in chromatograms similar to those presented in Fig. 6. Results are expressed as percentages of the amount of each steroid formed in the absence of etifoxine. Values are the mean (± SEM) of four independent experiments. *p<0.05; **p<0.01; ***p<0.001; NS, not statistically different from control (C).
Fig 9
Fig 9. Time-course of the conversion of tritiated pregnenolone ([3H]Δ5P) into radioactive 17-hydroxypregnenolone (17OH-Δ5P), dehydroepiandrosterone (DHEA), progesterone (P), dihydroprogesterone (DHP) and tetrahydroprogesterone (THP) by frog hypothalamic homogenates in the absence (○) or presence of 10-6 M etifoxine (●).
The values were calculated from the areas under the peaks in chromatograms similar to those presented in Fig. 6. Results are expressed as percentages of the amount of each steroid formed compared to the total amount of radiolabeled compounds resolved by HPLC analysis including [3H]Δ 5P. Values are the mean (± SEM) of four independent experiments. *p<0.05; **p<0.01; ***p<0.001 compared to respective control values; NS, not statistically different (one-way ANOVA followed by a post hoc Dunnett’s test).

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This work was partially supported by grants from Inserm (U413), IFRPM23/IRIB, University of Rouen and Region Haute-Normandie. Partial funding support was also provided by the pharmaceutical company BIOCODEX, which had no role in study design, data collection, analysis and interpretation, and writing of this manuscript.
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