Mitochondrial Diazepam Binding Inhibitor Receptor Research Articles

FGIN-1-27 Inhibits Melanogenesis by Regulating Protein Kinase A/cAMP-Responsive Element-Binding, Protein Kinase C-β, and Mitogen-Activated Protein Kinase Pathways.

FGIN-1-27 is a synthetic mitochondrial diazepam binding inhibitor receptor (MDR) agonist that has demonstrated pro-apoptotic, anti-anxiety, and steroidogenic activity in various studies. Here we report, for the first time, the anti-melanogenic efficacy of FGIN-1-27 in vitro and in vivo. FGIN-1-27 significantly inhibited basal and α-melanocyte-stimulating hormone (α-MSH)-, 1-Oleoyl-2-acetyl-sn-glycerol (OAG)- and Endothelin-1 (ET-1)-induced melanogenesis without cellular toxicity. Mushroom tyrosinase activity assay showed that FGIN-1-27 did not directly inhibit tyrosinase activity, which suggested that FGIN-1-27 was not a direct inhibitor of tyrosinase. Although it was not capable of modulating the catalytic activity of mushroom tyrosinase in vitro, FGIN-1-27 downregulated the expression levels of key proteins that function in melanogenesis. FGIN-1-27 played these functions mainly by suppressing the PKA/CREB, PKC-β, and MAPK pathways. Once inactivated, it decreased the expression of MITF, tyrosinase, TRP-1, TRP-2, and inhibited the tyrosinase activity, finally inhibiting melanogenesis. During in vivo experiments, FGIN-1-27 inhibited the body pigmentation of zebrafish and reduced UVB-induced hyperpigmentation in guinea pig skin, but not a reduction of numbers of melanocytes. Our findings indicated that FGIN-1-27 exhibited no cytotoxicity and inhibited melanogenesis in both in vitro and in vivo models. It may prove quite useful as a safer skin-whitening agent.

Neurosteroid allopregnanolone mediates anxiolytic effect of etifoxine in rats

Etifoxine (6-chloro-2-ethylamino-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride), a nonbenzodiazepine anxiolytic drug, potentiates GABA(A) receptor function perhaps through stimulation of neurosteroid biosynthesis. However, the exact mechanism of etifoxine action is not fully understood. In this study, we have assessed the possible role of GABAergic neurosteroid like allopregnanolone (ALLO) in the anxiolytic-like effect of etifoxine in rats using elevated plus maze test. Selective GABA(A) receptor agonist, muscimol, ALLO or neurosteroidogenic agents like progesterone, metyrapone or mitochondrial diazepam binding inhibitor receptor (MDR) agonist, FGIN 1-27 significantly heightened the etifoxine-induced anxiolysis. On the other hand, GABA(A) receptor antagonist, bicuculline or neurosteroid biosynthesis inhibitors like finasteride, indomethacin, trilostane or PBR antagonist, PK11195 significantly blocked the effect of etifoxine. Bilateral adrenalectomy did not influence anti-anxiety effect of etifoxine thereby ruling out contribution of adrenal steroids. Thus, our results provide behavioral evidence for the role of neurosteroids like ALLO in the anti-anxiety effect of etifoxine.

Essentiality of central GABAergic neuroactive steroid allopregnanolone for anticonvulsant action of fluoxetine against pentylenetetrazole-induced seizures in mice

Fluoxetine, a selective serotonin reuptake inhibitor, is known to increase the cortical content of allopregnanolone (ALLO) without altering the level of other neurosteroids. In contrast to the proconvulsant effect of many antidepressants, fluoxetine exhibits anticonvulsant effects. The present study was undertaken to examine the role of ALLO in the anticonvulsant action of fluoxetine against pentylenetetrazole (PTZ)-induced seizures in mice. Prior administration of GABA A receptor agonist muscimol or neurosteroid ALLO or progesterone, a precursor of ALLO or neurosteroidogenic drugs like FGIN 1–27, an agonist at the mitochondrial diazepam binding inhibitor receptor (MDR) or metyrapone, an 11β-hydroxylase inhibitor, significantly potentiated the anticonvulsant effect of fluoxetine. In contrast, the effect of fluoxetine was counteracted by inhibition of the neurosteroid biosynthesis using drugs like 5α-reductase inhibitor, finasteride; 3β-hydroxysteroid dehydrogenase inhibitor, trilostane; 3α-hydroxysteroid dehydrogenase inhibitor, indomethacin; MDR antagonist, PK 11195; or the GABA A receptor antagonist, bicuculline. Further, bilateral adrenalectomy had no significant effect on the anticonvulsant action of fluoxetine, suggesting negligible contribution from peripheral steroidogenesis. The anticonvulsant effect of fluoxetine was partially abolished in 5,7-DHT treated mice, indicating that the effect may also, in part, be dependent on serotonergic transmission. Thus, our data indicate that increased synthesis of ALLO in CNS is a major factor that ultimately leads to anticonvulsant effects of fluoxetine against PTZ-induced seizures.

Role of neuroactive steroid allopregnanolone in antipsychotic-like action of olanzapine in rodents.

Olanzapine increases brain allopregnanolone (ALLO) levels sufficiently to modulate neuronal activity by allosterically regulating GABAA receptors. Recently, we reported the antipsychotic-like profile of ALLO in rodents. The present study examined the hypothesis that olanzapine-induced elevation of endogenous neurosteroid ALLO is vital for its neuroleptic-like action. The conditioned avoidance response (CAR) and apomorphine-induced climbing behavioral paradigms were used in rodents. Administration of ALLO (1 microg, intracerebroventricular (i.c.v.)) or neurosteroidogenic agents such as the mitochondrial diazepam binding inhibitor receptor agonist, FGIN 1-27 (0.5 microg, i.c.v.) or the ALLO precursor, progesterone (10 mg/kg, i.p.) significantly potentiated olanzapine-induced blockade of CAR and apomorphine-induced climbing. In contrast, these agents failed to alter the antipsychotic-like effect of risperidone and haloperidol. On the other hand, inhibition of the endogenous biosynthesis of neurosteroids by the 3beta-hydroxysteroid dehydrogenase inhibitor, trilostane (30 mg/kg, i.p.), the 3alpha-hydroxysteroid oxidoreductase inhibitor, indomethacin (5 mg/kg, i.p.), or the GABAA receptor antagonist bicuculline (1 mg/kg, i.p.) and dehydroepiandrosterone sulfate (DHEAS) (1 mg/kg, i.p.) blocked the effect of olanzapine, but not of risperidone and haloperidol. Socially isolated animals, known to exhibit decreased brain ALLO and GABAA receptor functions, displayed a shortening in the muscimol-induced loss of righting reflex and an increased susceptibility to apomorphine-induced climbing. Administration of olanzapine, but not of haloperidol and risperidone, normalized the duration of muscimol-elicited loss of righting reflex. Although all three antipsychotics proved capable of antagonizing the apomorphine-induced climbing, a dose almost five times higher of olanzapine was required in socially isolated animals. The data obtained suggest that enhancement of the GABAergic tone plays a key role in the antipsychotic-like effect exerted by olanzapine in rodents, likely as a consequence of augmented levels of neuroactive steroids, in particular ALLO, in the brain. The present findings provide the first specific behavioral evidence in support of the hypothesis that neuroactive steroid ALLO- mediated GABAergic modulation is essential for the antipsychotic-like action of olanzapine.

Behavioral action of ethanol in Porsolt’s forced swim test: modulation by 3alpha-hydroxy-5alpha-pregnan-20-one

Ethanol is known to increase cortical and plasma content of GABAergic neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) which is responsible for some of its behavioral and electrophysiological effects. We have previously demonstrated the antidepressant like effect of 3α,5α-THP in mice. This study investigated the role of 3α,5α-THP in acute, chronic and withdrawal effects of ethanol using mouse forced swim test (FST) paradigm. While acute systemic ethanol (2 or 2.5 g/kg) administration exhibited an antidepressant like effect, its prolonged consumption produced tolerance to this effect and its withdrawal, on the other hand, elicited enhanced behavioral despair (depression). The antidepressant like effect of ethanol was potentiated by GABA A receptor agonist, muscimol (0.5 mg/kg, i.p.), 3α,5α-THP (0.5, 1 or 2 μg/mouse, i.c.v.) and by neurosteroidogenic drugs viz. selective serotonin reuptake inhibitor (SSRI), fluoxetine (5 or 20 mg/kg, i.p.), agonist at mitochondrial diazepam binding inhibitor receptor, FGIN 1-27 (0.5 or 1 μg/mouse, i.c.v.), or 11β-hydroxylase inhibitor, metyrapone (0.5 or 1 μg/mouse, i.c.v.) which are known to increase endogenous 3α,5α-THP content. Furthermore, inhibition of the endogenous neurosteroid biosynthesis by drugs like 5α-reductase inhibitor, finasteride (50 mg/kg, s.c.), 3β-hydroxysteroid dehydrogenase inhibitor, trilostane (30 mg/kg i.p.) or 3α-hydroxysteroid dehydrogenase inhibitor, indomethacin (5 mg/kg, i.p.) and GABA A receptor antagonist, bicuculline (1 mg/kg, i.p.) blocked the antidepressant like effect of ethanol. Withdrawal of ethanol from mice consuming it chronically displayed enhanced behavioral despair and elicited tolerance to antidepressant like action of acute ethanol (2.5, 3 or 3.5 g/kg). Moreover, sub-antidepressant doses (0.25 or 0.5 μg/mouse, i.c.v.) of 3α,5α-THP and fluoxetine (5 mg/kg, i.p.) but not imipramine (1 mg/kg, i.p.) reversed the depression associated with ethanol withdrawal indicating sensitization to their antidepressant action. Thus, 3α,5α-THP plays a pivotal role in the actions of ethanol and in the depression associated with ethanol withdrawal. These findings may be of potential ramification to contribute to the depression associated with alcoholism and its treatment using neurosteroids.

Subsensitivity to mitochondrial diazepam binding inhibitor receptor agonist FGIN-1-27-induced antiseizure effect in diazepam-withdrawn mice

We investigated the role of the mitochondrial diazepam binding inhibitor receptor (MDR) in diazepam-withdrawal seizure. In chronically vehicle-treated mice, the potent and selective MDR agonist FGIN-1-27 (N, N-di-n-hexyl 2-(4-fluorophenyl)indole-3-acetamide: 30 μg/mouse, i.c.v.) markedly increased the threshold for pentylenetetrazole (PTZ)-induced seizure. The antiseizure effect of FGIN-1-27 was blocked by pretreatment with the selective MDR antagonist PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide). In chronically diazepam-treated mice, the seizure threshold of PTZ was decreased during diazepam withdrawal, indicating withdrawal hyperexcitability. Interestingly, FGIN-1-27 (30 μg/mouse, i.c.v.) failed to increase the seizure threshold of PTZ in diazepam-withdrawn mice, in contrast to its effect in chronically vehicle-treated mice. These findings suggest that the sensitivity of MDR-mediated pathways in the brain may be decreased during diazepam withdrawal.

Chronic neurosteroid treatment prevents the development of morphine tolerance and attenuates abstinence behavior in mice

The effect of neurosteroids on the development of morphine tolerance and dependence was examined in mice. Development of tolerance to the antinociceptive effect of morphine sulfate (10 mg/kg, twice daily for 9 days) was measured in the tail-flick test and dependence was assessed from naloxone (2 mg/kg)-precipitated withdrawal jumps on day 10 of testing. Concomitant chronic administration of neurosteroids, allopregnanolone (0.5 mg/kg), pregnenolone sulfate (2 and 5 mg/kg) or dehydroepiandrosterone sulfate (2 and 5 mg/kg), followed by morphine (10 mg/kg) prevented the development of tolerance to the antinociceptive effect of morphine and suppressed the naloxone-precipitated withdrawal jumps. In contrast, dehydroepiandrosterone acetate (5 mg/kg) failed to modulate the morphine tolerance and dependence. The inhibitory effect was also seen upon concomitant administration of a neurosteroid precursor, progesterone (1–10 mg/kg), and a mitochondrial diazepam binding inhibitor receptor agonist, 4′-chlordiazepam (0.25–1 mg/kg), while an adrenocorticosteroid, hydrocortisone (1 and 10 mg/kg), failed to do so. However, acute treatment with these neurosteroids was not associated with any decrease in withdrawal jumping behavior in morphine-dependent mice. Neurosteroids themselves, at doses employed in the study, did not exert any effects on antinociception. These results support a role for neurosteroids in the development of tolerance to and dependence on morphine and suggest the potential utility of specific neuroactive steroids in its treatment.

Modulatory mechanisms of cyclic AMP-stimulated steroid content in rat brain cortex

The modulation of cyclic AMP dependent neurosteroidogenesis was studied in minces prepared from the cerebral cortex of adult rat. Forskolin or dibutyryl-cyclic AMP enhanced pregnenolone and progesterone production in a time and dose-dependent manner. The forskolin effect was mimicked by the cyclic AMP phosphodiesterase inhibitor isobutyl-methyl-xanthine, but not by the adenylate cyclase inactive forskolin analogue 1,9,dideoxy-forskolin. 4′-Chloro-diazepam, a high affinity ligand for the mitochondrial diazepam binding inhibitor (DBI) receptor, also elicited a time dependent increase in steroidogenesis. The forskolin and the 4′-chloro-diazepam stimulated pregnenolone increase was prevented by preexposing the rat brain cortical minces to 1-(2-chlorophenyl)- N-(1-methyl-propyl)-3-isoquinoline carboxamide (PK 11195), a high affinity ligand for the mitochondrial DBI receptor endowed with antagonistic properties. The protein synthesis inhibitor cycloheximide prevented the forskolin and 4′-chloro-diazepam stimulation of pregnenolone formation. In brain cortical minces of adrenalectomised/ orchiectomised rats dibutyryl-cyclic AMP increased both pregnenolone and progesterone formation, while forskolin only increased progesterone. These data show that cyclic AMP enhances brain steroidogenesis by acting on a labile protein substrate which interacts with the mitochondrial DBI receptor.

The pharmacology of neurosteroidogenesis

In adrenal cortex and other steroidogenic tissues including glial cells, the conversion of cholesterol into pregnenolone is catalyzed by the cytochrome P450 scc located in the inner mitochondrial membrane. A complex mechanism operative in regulating cholesterol access to P450 scc limits the rate of pregnenolone biosynthesis. Participating in this mechanism are DBI (diazepam binding inhibitor), an endogenous peptide that is highly expressed in steroidogenic cells and some of the DBI processing products including DBI 17–50 (TTN). DBI and TTN activate steroidogenesis by binding to a specific receptor located in the outer mitochondrial membrane, termed mitochondrial DBI receptor complex (MDRC). MDRC is a hetero-oligomeric protein: only the subunit that includes the DBI and benzodiazepine (BZD) recognition sites has been cloned. Several 2-aryl-3-indoleacetamide derivatives (FGIN-1-X) with highly selective affinity (nM) for MDRC were synthesized which can stimulate steroidogenesis in mitochondrial preparations. These compounds stimulate adrenal cortex steroidogenesis in hypophysectomized rats but not in intact animals. Moreover, this steroidogenesis is inhibited by the isoquinoline carboxamide derivative PK 11195, a specific high affinity ligand for MDRC with a low intrinsic steroidogenic activity. Some of the FGIN-1-X derivatives stimulate brain pregnenolone accumulation in adrenalectomized-castrated rats. The FGIN-1-X derivatives that increase brain pregnenolone content, elicit antineophobic activity and antagonize punished behavior in the Vogel conflict test in rats. These actions of FGIN-1-X are resistant to inhibition by flumazenil, a specific inhibitor of BZD action in GABA A receptors but are antagonized by PK 11195, a specific blocker of the steroidogenesis activation via MDRC stimulation. It is postulated that the pharmacological action of FGIN-1-X depends on a positive modulation of the GABA action on GABA A receptors mediated by the stimulation of brain neurosteroid production.

Stimulation of brain pregnenolone synthesis by mitochondrial diazepam binding inhibitor receptor ligands in vivo.

Evidence that neurosteroids are potent modulators of the action of GABA at GABAA receptors has prompted the investigation of the mechanism that controls brain neurosteroid synthesis by glial cell mitochondria in vivo. In vitro studies suggest that the interaction of the diazepam binding inhibitor (DBI)--a polypeptide that is abundant in steroidogenic cells--with glial mitochondrial DBI receptors (MDRs) is a crucial step in the physiological regulation of neurosteroid biosynthesis. MDRs bind 4'-chlorodiazepam (4'-CD), N,N-di-n-hexyl-2-(4-fluorophenyl)-indol-3-acetamide (FGIN-1-27), and the isoquinoline carboxamide PK 11195 with high affinity, and these ligands have been used to investigate whether the stimulation of glial MDRs increases brain pregnenolone production in vivo. Adrenalectomized and castrated (A-C) male rats (to eliminate peripheral sources of pregnenolone) were pretreated with trilostane (to prevent pregnenolone metabolism to progesterone), and the pregnenolone content in brain regions dissected after fixation with a 0.8-s exposure to microwave irradiation focused to the head was determined by HPLC followed by specific radioimmunoassay. The forebrain and cerebellum of A-C rats contained 4-7 ng of pregnenolone/g of tissue, and the olfactory bulb contained 10-14 ng/g. These concentrations of brain pregnenolone are only 30-40% lower than those of sham-operated rats. In contrast, the plasma pregnenolone content of sham-operated rats was 2-3 ng/ml, but it was only 0.15-0.20 ng/ml in the plasma of A-C rats. In A-C rats, treatment with the MDR ligands 4'-CD and FGIN-1-27 increased the pregnenolone content in the brain but failed to change the plasma or peripheral tissue content of this steroid. The effect of 4'-CD on brain pregnenolone content was maximal (70-100% increase) at the dose of 18 mumol/kg, 5-10 min after intravenous injection. The effect of oral administration of FGIN-1-27 on brain pregnenolone content was maximal (80-150% increase) at doses of 400-800 mumol/kg and peaked at approximately 1 h. That this effect of FGIN-1-27 was mediated by the MDR was documented by pretreatment with the MDR partial agonist PK 11195 (100 mumol/kg, i.p.). PK 11195 did not affect basal brain pregnenolone content but prevented the accumulation of brain pregnenolone induced by FGIN-1-27. FGIN-1-27 and 4'-CD failed to increase the brain concentration of dehydroepiandrosterone in A-C rats. These data suggest that glial cell MDRs play a role in neurosteroid biosynthesis in vivo.

ACTH-induced mitochondrial DBI receptor (MDR) and diazepam binding inhibitor (DBI) expression in adrenals of hypophysectomized rats is not cause-effect related to its immediate steroidogenic action

Diazepan binding inhibitor (DBI) is a 10-kDa polypeptide that is enriched in steroidogenic cells such as adrenocortical, Leydig, and glial cells. In these cells, DBI and some of its processing products bind to the mitochondrial DBI receptor (MDR), located on the outer mitochondrial membrane, and stimulate pregnenolone formation by facilitating cholesterol access to the inner mitochondrial membrane where the cytochrome P-450 side chain cleavage enzyme is located. To determine whether the ACTH-induced increase in adrenal steroidogenesis occurs via changes in DBI and MDR expression the adrenal content of DBI-like immunoreactivity (DBI-LI), the MDR density, and the expression of mRNAs encoding for DBI and MDR were studied in hypophysectomized rats treated with vehicle or ACTH. After 9 days from the hypophysectomy, the levels of DBI-like immunoreactivity (DBI-LI) and DBI-mRNA declined to approximately 20% of their normal value; in contrast MDR-density and MDR-mRNA levels were reduced by 50–60% and were associated to a similar decrease in the activity of type A monoamine oxidase, a marker for mitochondrial proteins. Prolonged administration of ACTH-R (ACTH in saline containing 16% gelatin, 15 U/kg/day, from day 7 after surgery) to hypophysectomized rats, completely restored DBI and MDR adrenal expression to values similar to those of sham-operated rats. Our results indicate that ACTH, probably acting at the transcriptional level, is required for the normal expression of DBI and MDR in adrenal cortex. Changes in DBI and MDR expression after ACTH administration were not temporally related to the immediate steroidogenesis induced by ACTH, and may reflect its long-term trophic action on adrenocortical cells.

Diazepam-binding inhibitor (DBI)-processing products, acting at the mitochondrial DBI receptor, mediate adrenocorticotropic hormone-induced steroidogenesis in rat adrenal gland.

Diazepam-binding inhibitor (DBI) is a 9-kDa polypeptide that colocalizes in glial, adrenocortical, and Leydig cells with the mitochondrial DBI receptor (MDR). By binding with high affinity to the MDR, DBI and one of its processing products--DBI-(17-50)--regulate pregnenolone synthesis and have been suggested to participate in the immediate activation of adrenal steroidogenesis by adrenocorticotropic hormone (ACTH). In adrenals of hypophysectomized rats (1 day after surgery), ACTH failed to acutely affect the amount of adrenal DBI and the density of MDR but increased the rate of DBI processing, as determined by the HPLC profile of DBI-(17-50)-like immunoreactivity. The similar latency times for this effect and for ACTH stimulation of adrenal steroidogenesis suggest that the two processes are related. The ACTH-induced increase in both adrenal steroidogenesis and rate of DBI processing were completely inhibited by cycloheximide; this result suggests the requirement for the de novo synthesis of a protein with a short half-life, probably an endopeptidase. This enzyme, under the influence of ACTH, may activate formation of a DBI-processing product that stimulates steroidogenesis via the MDR. In support of this hypothesis is the demonstration that in hypophysectomized rats the MDR antagonist PK 11195 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide completely inhibited the adrenal steroidogenesis stimulated by ACTH and by the high-affinity MDR ligand 4'-chlorodiazepam.

Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.

The C6-2B glioma cell line, rich in mitochondrial receptors that bind with high affinity to benzodiazepines, imidazopyridines, and isoquinolinecarboxamides (previously called peripheral-type benzodiazepine receptors), was investigated as a model to study the significance of the polypeptide diazepam binding inhibitor (DBI) and the putative DBI processing products on mitochondrial receptor-regulated steroidogenesis. DBI and its naturally occurring fragments have been found to be present in high concentrations in C6-2B glioma cells, to compete against specific isoquinolinecarboxamide or 4'-chlorodiazepam binding to mitochondrial recognition sites with high affinity, and to stimulate mitochondrial pregnenolone formation. These data suggest that this cell type may express both the receptor and the putative agonist ligand to regulate steroidogenesis. Therefore, we propose to term this mitochondrial receptor MDR (mitochondrial DBI receptor) to indicate its responsiveness to DBI in steroid biosynthesis. In the present work, we show that mitochondria of C6-2B cells convert (22R)-22-hydroxycholesterol to pregnenolone by a mechanism blocked by aminoglutethimide. Immunoblotting confirmed the presence of relatively high levels of cytochrome P-450 cholesterol side-chain-cleavage enzyme in C6-2B cell mitochondria. Furthermore, isoquinolinecarboxamide binding sites associated with the 18-kDa mitochondrial polypeptide subunit of the MDR are abundant in C6-2B glioma cell mitochondria (Bmax approximately 30 pmol/mg protein) and are coupled to the regulation of steroid biosynthesis. Occupancy of MDRs with nanomolar concentrations of the naturally occurring polypeptide, DBI, as well as its naturally occurring processing product tetratriacontaneuropeptide [DBI-(17-50)] increases pregnenolone formation. Clonazepam and octadecaneuropeptide [DBI-(33-50)], which exhibit a higher affinity for gamma-aminobutyric acid type A receptors but a low affinity for MDR, were ineffective in stimulating pregnenolone synthesis. These findings provide evidence that C6-2B cells exhibit a significant steroidogenic activity which resembles that found in peripheral endocrine organs and they suggest that MDRs and DBI are involved in the regulation of glial cell steroidogenesis.