Alessandro Guidotti

Diazepam binding inhibitor (DBI): A peptide with multiple biological actions

Diazepam binding inhibitor (DBI) is a 9-kD polypeptide that was first isolated in 1983 from rat brain by monitoring its ability to displace diazepam from the benzodiazepine (BZD) recognition site located on the extracellular domain of the type A receptor for gamma-aminobutyric acid (GABAA receptor) and from the mitochondrial BZD receptor (MBR) located on the outer mitochondrial membrane. In brain, DBI and its two major processing products [DBI 33-50, or octadecaneuropeptide (ODN) and DBI 17-50, or triakontatetraneuropeptide (TTN)] are unevenly distributed in neurons, with the highest concentrations of DBI (10 to 50 microMs) being present in the hypothalamus, amygdala, cerebellum, and discrete areas of the thalamus, hippocampus, and cortex. DBI is also present in specialized glial cells (astroglia and Bergmann glia) and in peripheral tissues. In the periphery, the highest concentration of DBI occurs in cells of the zona glomerulosa and fasciculata of the adrenal cortex and in Leydig cells of the testis; interestingly, these are the same cell types in which MBRs are highly concentrated. Stimulation of MBRs by appropriate ligands (including DBI and TTN) facilitates cholesterol influx into mitochondria and the subsequent formation of pregnenolone, the parent molecule for endogenous steroid production; this facilitation occurs not only in peripheral steroidogenic tissues, but also in glial cells, the steroidogenic cells of the brain. Some of the steroids (pregnenolone sulfate, dehydroepiandrosterone sulfate, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one) produced in brain (neurosteroids) function as potent (with effects in the nanomolar concentration range) positive or negative allosteric modulators of GABAA receptor function. Thus, accumulating evidence suggests that the various neurobiological actions of DBI and its processing products may be attributable to the ability of these peptides either to bind to BZD recognition sites associated with GABAA receptors or to bind to glial cell MBRs and modulate the rate and quality of neurosteroidogenesis. The neurobiological effects of DBI and its processing products in physiological and pathological conditions (hepatic encephlopaty, depression, panic) concentrations may therefore be explained by interactions with different types of BZD recognition site. In addition, recent reports that DBI and some of its fragments inhibit (in nanomolar concentrations) glucose-induced insulin release from pancreatic islets and bind acyl-coenzyme A with high affinity support the hypothesis that DBI isa precursor of biologically active peptides with multiple actions in the brain and in peripheral tissues.

A brain octadecaneuropeptide generated by tryptic digestion of DBI (diazepam binding inhibitor) functions as a proconflict ligand of benzodiazepine recognition sites

An octadecaneuropeptide (ODN) produced by the tryptic digestion of DBI was purified and sequenced and its activity on the Vogel test determined. In vitro ODN displaces 3H-diazepam from specific brain recognition sites and injected intraventricularly in thirsty rats facilitates the onset of behavioral inhibition elicited by punishment. The alpha-amide derivative of ODN is devoid of either action. Evidence is presented suggesting that DBI sequence includes at least two replicas of ODN or one replica of ODN and a fragment with similar if not identical amino acid sequence but identical biological activity.

Abusive stimulation of excitatory amino acid receptors: a strategy to limit neurotoxicity.

Glutamate is an important excitatory amino acid at many central nervous system synapses. After its release from presynaptic nerve terminals, glutamate transiently binds to specific neuronal membrane receptors, which transduce its signal by the generation of intracellular second messengers before being rapidly cleared from the synapse. However, during ischemia, the glutamate concentration at synapses surrounding the focal lesion can be increased for sustained periods of time, resulting in abusive stimulation of glutamate receptors that can eventually be neurotoxic. To develop drugs capable of selectively blocking the pathological effects of glutamate in neurons surrounding ischemic lesions while leaving the physiological actions of glutamate in nonlesioned areas of the brain unaffected, it is essential to delineate glutamate‐induced intracellular events that are specific to receptor abuse. This article describes the intracellular sequelae of physiological and pathological glutamate receptor activation and suggests potential targets for such receptor abuse‐dependent antagonists (RADAs).— Manev, H.; Costa, E.; Wroblewski, J. T.; Guidotti, A. Abusive stimulation of excitatory amino acid receptors: a strategy to limit neurotoxicity: FASEB J. 4: 2789‐2797; 1990.

Stimulation of Brain Pregnenolone Synthesis by Mitochondrial Diazepam Binding Inhibitor Receptor Ligands In Vivo

Abstract: 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 shamoperated 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 μmol/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 μmollkg and peaked at ∼ 1 h. That this effect of FGIN‐1–27 was mediated by the MDR was documented by pre‐treatment with the MDR partial agonist PK 11195 (100 μmol/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 dehydre epiandrosterone in A‐C rats. These data suggest that glial cell MDRs play a role in neurosteroid biosynthesis in vivo.

Endogenous benzodiazepine receptor ligands in human and animal hepatic encephalopathy

Abstract: The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalopathy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and purification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were carried out using both radioreceptor binding assays and radioimmunoassays with anti‐benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and desmethyldiazepam, based on elution profiles and anti‐benzodiazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained ∼ 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver disease. The levels of brain benzodiazepine receptor ligand compounds were also increased ∼ 10‐fold in rats suffering from fulminant hepatic failure, but not in rats with portacaval shunts, a model of chronic hepatic disease. The increased concentrations of these substances could be behaviorally significant and may contribute to the pathogenesis of hepatic encephalopathy.

Isolation and Characterization of a Rat Brain Triakontatetraneuropeptide, a Posttranslational Product of Diazepam Binding Inhibitor: Specific Action at the Ro 5‐4864 Recognition Site

Abstract: This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17‐50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octa‐decaneuropeptide, DBI 33‐50) antibodies coupled to CNBr‐Sepharose 4B followed by two reverse‐phase HPLC steps. The amino acid sequence of the purified peptide is: Thr‐Gln‐Pro‐Thr‐Asp‐Glu‐Glu‐Met‐Leu‐Phe‐Ile‐Tyr‐Ser‐His‐Phe‐Lys‐Gln‐Ala‐Thr‐Val‐Gly‐Asp‐Val‐Asn‐Thr‐Asp‐Arg‐Pro‐Gly‐Leu‐Leu‐Asp‐Leu‐Lys. Synthetic TTN injected intra‐cerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific “peripheral” benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the “central” BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5‐4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 μM. TTN also enhances picrotoxinin inhibition of γ‐aminobutyric acid (GABA)‐stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at “Ro 5‐4864 preferring” BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor‐mediated effects.

Distribution and metabolism of muscimol in the brain and other tissues of the rat

Abstract Using 3H-methylene-muscimol of high specific activity (12.1 Ci/mmol, NEN, MA), a simple, ion exchange Chromatographic method was developed to study muscimol distribution in rats. After intravenous injection muscimol entered the brain and distributed unevenly to various brain regions. The brain regions with the highest muscimol concentrations were the substantia nigra, the colliculi and the hypothalamus. The muscimol recovered in brain after an intravenous injection of 8 μmol/kg was only 0.02% of the amount injected. Its concentration in brain (approximately 200 pmol/g tissue) was sufficient to explain some of its GABA mimetic actions. After intravenous injection, muscimol disappeared very rapidly from blood. Its disappearance was paralleled by an increase in plasma levels of radioactive muscimol metabolites. When the injection of muscimol was preceded by pretreatment with AOAA (a blocker of transamination activity) the tissue concentrations of muscimol were several times greater than those of untreated rats: moreover, muscimol metabolites in blood and brain were reduced. Brain levels of muscimol were not changed after a pretreatment with pargyline or with 2-dieth-ylaminoethyl-2, 2-diphenylvalerate, hydrochloride (SKF 525A). Thus, transamination appears to be one of the major pathways for the metabolism of muscimol.

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 P450scc located in the inner mitochondrial membrane. A complex mechanism operative in regulating cholesterol access to P450scc 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 GABAA 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 GABAA receptors mediated by the stimulation of brain neurosteroid production.

Location and characterization of opiate receptors regulating pituitary secretion

Abstract The site at which opiate agonists and antagonists act to alter secretion of prolactin, growth hormone and luteinizing hormone as well as the pharmacological specificity of the opiate receptors mediating these effects were examined in rats. Injection of β-endorphin but not a 10 fold higher dose of the non opiate peptide β-endorphin, increased release of prolactin and growth hormone in male rats while inhibiting luteinizing hormone release in ovariectomized, estrogen primed female rats. Prior treatment with naltrexone i.p. blocked these responses. Injection of naltrexone into the hypothalamus lowered prolactin release. In rats with a surgically formed hypothalamic island systemic administration of morphine or naltrexone altered prolactin release in the same manner as was observed in intact animals. In contrast no effects of β-endorphin or naltrexone were observed on the spontaneous secretion of prolactin in vitro . In addition β-endorphin did not alter the inhibition of prolactin release produced by apomorphine in vitro . The ED50 for stimulation of prolactin release following intraventricular administration of β-endorphin or the synthetic enkephalin analog FK 33-824 was the same, approximately 0.1 ng/rat. However FK 33-824 at 0.2 ng/rat was able to produce much greater analgesia and catatonia than β-endorphin. The metabolism and distribution of β-endorphin was examined but did not account for these differential effects. These results indicate that opiate agonists and antagonists can act at the hypothalamic but not the anterior pituitary level to alter release of prolactin, growth hormone and luteinizing hormone. In addition the data suggest that the opiate receptors mediating release of prolactin may have a different pharmacological specificity from those involved with analgesia and catatonia.

Isolation, purification and partial sequence of a neuropeptide (diazepam-binding inhibitor) precursor of an anxiogenic putative ligand for benzodiazepine recognition site

Diazepam binding inhibitor (DBI), a brain neuropeptide putative ligand for benzodiazepine binding sites, has been isolated and purified to homogeneity. This compound, like the anxiogenic beta-carbolines, injected intracerebroventricularly facilitates shock-induced suppression of drinking in thirsty rats. Cyanogen bromide (CNBr) cleavage of DBI produces three peptide fragments: the carboxy terminal fragment (F3 approximately equal to 1800 mol.wt.) and an intermediate fragment (F2 approximately equal to 3200 mol.wt.) are inactive, whereas the fragment that contains the amino terminus (F1 approximately equal to 6500 mol. wt.) facilitates punishment inhibition of operant behavior in rat. These data suggest that the F1 peptide contains the active sequence. The latter might be the natural effector of benzodiazepine recognition sites while DBI could be a polyprotein functioning as the precursor of the putative endogenous ligand of the benzodiazepine recognition site.