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A Guidotti

Publications and source records attributed to A Guidotti.

At least 163 records · Page 9Linked to original sources
PubMed1988

Molecular characterization and mitochondrial density of a recognition site for peripheral-type benzodiazepine ligands.

In a previous report, mitochondria were proposed as a subcellular structure where recognition sites for peripheral benzodiazepine ligands are located in adrenal glands. The present study examines the subcellular distribution of specific binding sites for PK 11195 in eight tissues and compares the relative densities of these binding sites in mitochondrial-enriched fractions with the relative activities of two mitochondrial marker enzymes. In all eight tissues examined, PK 11195 binding sites were found to subfractionate in a manner nearly identical to that of the mitochondrial enzyme succinate dehydrogenase. The subcellular distribution patterns of specific PK 11195 binding sites were unrelated to the distribution patterns of marker enzymes for plasma membranes, lysosomes, or endoplasmic reticulum. Scatchard analyses of mitochondrial fractions from all eight tissues demonstrated a greater than 100-fold difference in the densities of PK 11195 binding sites, the extremes being 140 and 1 pmol/mg of protein in adrenal and brain tissues, respectively. There was no correlation between the relative density of PK 11195 binding sites and the specific activities of succinate dehydrogenase and cytochrome c oxidase. These results suggest that the density of peripheral-type benzodiazepine receptors in mitochondria is tissue dependent and apparently regulated independently of the mechanisms by which these two mitochondrial enzymes are expressed or function. The photoaffinity probe PK 14105 was used to photolabel the peripheral-type benzodiazepine binding sites of mitochondrial fractions prepared from the eight tissues. In all preparations, a 17,000-Da polypeptide is specifically labeled as determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Thus, it appears that the protein recognition site for isoquinoline carboxamides of peripheral-type benzodiazepine receptor complexes is similar in all mitochondrial preparations.

Affinity Labels↗
PubMed1987

Co-localization and co-release of GABA and putative allosteric modulators of GABA receptor.

Diazepam binding inhibitor (DBI) belongs to a family of newly discovered neuropeptides that, when acting on the benzodiazepine/beta-carboline recognition site, provide an allosteric modulation of the function of GABAA receptor. The molecular size of DBI (10K Da) and its amino acid sequence characteristics are compatible with the view that this polypeptide can function as a precursor of smaller biologically active neuropeptides. In neurons of the cerebral cortex of the neonatal rat, in primary culture, DBI coexists with at least 4 different processing products. These peptides immunoreact with an antiserum directed against a biologically active octadecaneuropeptide (ODN) amino acid sequence of which (QATVGDVNTDRPGLLDLK) is included in the middle portion of the amino acid sequence of DBI. One of the immunoreactive peptides extracted from neurons has a retention time in high pressure liquid chromatography (HPLC) identical to that of synthetic ODN. Double immunofluorescence staining of the cultured neurons with glutamic acid decarboxylase (GAD) and antibodies for ODN indicates that ODN and ODN-like peptides are localized with GABA in 58% of the GAD-positive neurons. Moreover, the proportion of the neuronal stores of GABA, ODN, DBI-like peptides and DBI that are released together following depolarization with veratridine is similar. These experiments provide evidence to suggest that ODN, ODN-like peptides derived from DBI, might participate as putative neuromodulators of physiological significance in changing the probability that a quantum of GABA opens specific chloride (Cl-) channels located on post-synaptic cell membranes.

Amino Acid Sequence↗
PubMed1987

Ganglioside inhibition of glutamate-mediated protein kinase C translocation in primary cultures of cerebellar neurons.

In primary cultures of cerebellar granule cells, protein kinase C (PKC) translocation and activation can be triggered by the stimulation of excitatory amino acid neurotransmitter receptors. Glutamate evokes a dose-related translocation of 4-beta-[3H]phorbol 12,13-dibutyrate ([3H]-P(BtO)2) binding sites from the cytosol to the neuronal membrane and stimulates the incorporation of 32P into a number of membrane proteins, particularly protein bands in the range of 80, 50, and 40 kDa. The glutamate-evoked PKC translocation is Mg2+ sensitive, is prevented by 2-amino-5-phosphonovalerate and phencyclidine, is not inhibited by nitrendipine (a voltage-dependent Ca2+-channel blocker) but is abolished by the removal of Ca2+ from the incubation medium, suggesting that glutamate-mediated Ca2+ influx is operative in the redistribution of PKC. Exposure of granule cells to the gangliosides trisialosylgangliotetraglycosylceramide (GT1b) or monosialosylgangliotetraglycosylceramide (GM1) inhibits the translocation and activation of PKC evoked by glutamate. These glycosphingolipids fail to interfere with glutamate binding to its high-affinity recognition site or with the [3H]P(BtO)2 binding, nor do they affect the Ca2+ influx. These gangliosides may prevent PKC translocation by interfering with the PKC binding to the neuronal membrane phosphatidylserine.

Animals↗
PubMed1987

Protracted treatment with diazepam increases the turnover of putative endogenous ligands for the benzodiazepine/beta-carboline recognition site.

DBI (diazepam-binding inhibitor) is a putative neuromodulatory peptide isolated from rat brain that acts on gamma-aminobutyric acid-benzodiazepine-Cl- ionophore receptor complex inducing beta-carboline-like effects. We used a cDNA probe complementary to DBI mRNA and a specific antibody for rat DBI to study in rat brain how the dynamic state of DBI can be affected after protracted (three times a day for 10 days) treatment with diazepam and chlordiazepoxide by oral gavage. Both the content of DBI and DBI mRNA increased in the cerebellum and cerebral cortex but failed to change in the hippocampus and striatum of rats receiving this protracted benzodiazepine treatment. Acute treatment with diazepam did not affect the dynamic state of brain DBI. An antibody was raised against a biologically active octadecaneuropeptide (Gln-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys ) derived from the tryptic digestion of DBI. The combined HPLC/RIA analysis of rat cerebellar extracts carried out with this antibody showed that multiple molecular forms of the octadecaneuropeptide-like reactivity are present and all of them are increased in rats receiving repeated daily injections of diazepam. It is inferred that tolerance to benzodiazepines is associated with an increase in the turnover rate of DBI, which may be responsible for the gamma-aminobutyric acid receptor desensitization that occurs after protracted benzodiazepine administration.

Animals↗
PubMed1987

Subcellular location and neuronal release of diazepam binding inhibitor.

Diazepam binding inhibitor (DBI), a peptide located in CNS neurons, blocks the binding of benzodiazepines and beta-carbolines to the allosteric modulatory sites of gamma-aminobutyric acid (GABAA) receptors. Subcellular fractionation studies of rat brain indicate that DBI is compartmentalized. DBI-like immunoreactivity is highly enriched in synaptosomes obtained by differential centrifugation in isotonic sucrose followed by a Percoll gradient. In synaptosomal lysate, DBI-like immunoreactivity is primarily associated with synaptic vesicles partially purified by differential centrifugation and continuous sucrose gradient. Depolarization induced by high K+ levels (50 mM) or veratridine (50 microM) released DBI stored in neurons of superfused slices of hypothalamus, hippocampus, striatum, and cerebral cortex. The high K+ level-induced release is Ca2+ dependent, and the release induced by veratridine is blocked by 1.7 microM tetrodotoxin. Depolarization released GABA and Met5-enkephalin-Arg6-Phe7 together with DBI. DBI is also released by veratridine depolarization, in a tetrodotoxin-sensitive fashion, from primary cultures of cerebral cortical neurons, but not from cortical astrocytes. Depolarization fails to release DBI from slices of liver and other peripheral organs. These data support the view that DBI may be released as a putative neuromodulatory substance from rat brain neurons.

Animals↗
PubMed1987

Effect of gamma-aminobutyric acidA receptor agonists and antagonists on the release of enkephalin-containing peptides from dog adrenal gland.

Chromaffin cells of the adrenal medulla are known to store and release catecholamines, Met5-enkephalin (ME)-like peptides and gamma-aminobutyric acid (GABA). The present study documents that stimulation of GABAA receptors located on chromaffin cell membranes of canine adrenal glands, eliciting depolarization of chromaffin cell membranes, modulates the responsiveness of chromaffin cells to splanchnic nerve stimulation. 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (0.143 mmol/2 ml/min), a selective GABAA receptor agonist infused into the aortic pouch, increases the release of ME-like peptides and catecholamines into the adrenal effluent blood. Prior infusion into the aortic pouch of the GABAA receptor blocker, bicuculline (0.05 mmol/2 ml/min), prevents the 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol-elicited release of both substances. A stoichiometric relationship exists between the release of both substances; 1 nmol/ml of plasma of catecholamines was coreleased with 2 pmol/ml of plasma of ME-like peptides. The chromatographic profile on a Sephadex G-75 column indicates that, after injection of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, various MW forms of ME-like peptides are released into the adrenal effluent blood. A similar profile for the release of ME-like peptides was obtained when electrical stimulation (10 V/6 Hz) of the splanchnic nerve was used as a stimulus. These data suggest that direct stimulation of GABAA receptors causes depolarization of chromaffin cell membranes by a burst of Cl- channel opening and triggers neurotransmitter release.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1987

Excitatory amino acid receptors coupled with guanylate cyclase in primary cultures of cerebellar granule cells.

Primary cultures of cerebellar granule cells have been used in pharmacologically and functionally characterizing excitatory amino acid recognition sites coupled with guanylate cyclase. When granule cells were incubated in physiological culture conditions (Locke's solution, pH 7.4), only kainate and, to a lesser extent, L-glutamate increased cyclic GMP (cGMP) levels. Under these conditions, L-aspartate, N-methyl-D-aspartate (NMDA), and quisqualate were inactive. When granule cells were incubated in the absence of extracellular Mg2+ or in the presence of the depolarizing agent veratrine, L-glutamate, L-aspartate, and NMDA became as effective as kainate in enhancing cGMP formation. The action of kainate was preferentially antagonized by 2,3-cis-piperidindicarboxylate, whereas the action of L-glutamate was preferentially antagonized by (+/-)2-amino-5-phosphonovalerate. These data suggest that 2 different excitatory amino acid recognition sites (activated by kainate or by L-glutamate, L-aspartate, and NMDA, respectively) are coupled with guanylate cyclase in primary cultures of cerebellar granule cells: While the coupling of the recognition site for kainate with guanylate cyclase operates under resting conditions and in the presence of Mg2+, the coupling of the recognition site for L-glutamate, L-aspartate, and NMDA with guanylate cyclase requires depolarizing conditions or the absence of extracellular Mg2+.

Amino Acids↗
PubMed1987

Coexistence of GABA receptors and GABA-modulin in primary cultures of rat cerebellar granule cells.

GABA-modulin (GM), a basic polypeptide purified from rat brain synaptosomes, which is an allosteric inhibitor of GABA recognition sites, has been detected in primary cultures of cerebellar interneurons enriched in granule cells by immunohistochemistry, using a specific antibody raised in rabbit injected with GM purified from rat brain synaptosomes. In these cultures, GM is expressed by the granule cells, which are postsynaptic to GABAergic interneurons, but not by glial cells. In rat cerebellar sections anti-GM antiserum intensely strains the granular cell layer and Purkinje cell dendrites and cell bodies. GM has been purified from the cerebellar granule cell cultures and appears to be identical under biochemical, immunological, and functional criteria to authentic GM purified from rat brain synaptosomes. Granule cell cultures devoid of GABAergic neurons contain the GABA/BZ/Cl- receptor complex; in fact, intact cell monolayers, incubated in physiological buffer at 25 degrees C, express 3H-muscimol and 3H-flunitrazepam binding sites, which are comparable to the sites detected in cell membrane preparations and which modulate each other reciprocally. It is concluded that GM might participate in the supramolecular organization of the GABA receptor complex, perhaps functioning as a modulator of this receptor protein.

Animals↗
PubMed1987

Studies of a brain polypeptide functioning as a putative endogenous ligand to benzodiazepine recognition sites in rats selectively bred for alcohol related behavior.

The brain content of Diazepam Binding Inhibitor (DBI), its cell location and that of its specific mRNA were studied immunohistochemically and by in situ hybridization. Various strains of rats were genetically selected for their alcohol tolerance and the above mentioned brain parameters were studied before and after chronic ethanol consumption. The DBI like immunoreactivity (DBI-LI) was found to be located in selected neuronal population and in non-neuronal cells. The DBI-mRNA was located in brain areas where DBI is abundant. It was immunochemically determined that the DBI content was increased in cerebellum and in hypothalamus of alcohol preferring rats after chronic ethanol consumption. DBI content was compared in the cerebellum of rats genetically selected for different alcohol sensitivity and it was significantly higher on the ethanol sensitive (ANT) rat strain.

Alcoholism↗
PubMed1986

A diazepam binding inhibitor (DBI)-like neuropeptide is detected in human brain.

Diazepam binding inhibitor (DBI), a 11,000 MW neuropeptide, which coexists with GABA and elicits proconflict responses in the rat, has been purified and partially sequenced from rat brain. We now report purification and characterization of a DBI-like neuropeptide from human brain. Its molecular weight and pharmacological profile is identical to that of rat DBI but differs in the amino acid composition and immunologically. The tryptic fragments of human DBI differ from rat DBI in the HPLC elution profile and in the amino acid sequence. Using high affinity specific human DBI antibodies, the distribution of DBI-like immunoreactivity in bioptic samples of human brain appeared to be similar to that of DBI found in rat brain. DBI-like immunoreactivity was also found in spinal fluid of human volunteers. The cerebrospinal fluid content of this peptide might be used as a probe to study whether spinal fluid DBI content changes in neuropsychiatric disorders.

Amino Acid Sequence↗
PubMed1986

Diazepam-binding inhibitor. A brain neuropeptide present in human spinal fluid: studies in depression, schizophrenia, and Alzheimer's disease.

Diazepam-binding inhibitor is a novel peptide purified to homogeneity from rat and human brain. Diazepam-binding inhibitor is present, though not exclusively, in gamma-aminobutyric acid (GABA)-containing neurons where it is believed to inhibit GABAergic neurotransmission mediated by GABA by binding to the benzodiazepine-GABA receptor complex. Since an impairment of central GABAergic tone has been postulated to be associated with a number of neuropsychiatric disorders, we measured human diazepam-binding inhibitor immunoreactivity in the cerebrospinal fluid (CSF) of patients suffering from endogenous depression, schizophrenia, and dementia of the Alzheimer's type. Patients with major depression had significantly higher concentrations of human diazepam-binding inhibitor immunoreactivity in CSF when compared with age- and sex-matched normal volunteers, while no difference in CSF diazepam-binding inhibitor immunoreactivity was found in schizophrenics or patients with dementia of the Alzheimer's type when compared with controls. The possibility is discussed that the increased CSF human diazepam-binding inhibitor immunoreactivity observed in depressed patients may represent a functional disinhibition of GABAergic neurotransmission associated with depression.

Adult↗
PubMed1986

Cloning and expression of cDNA for human diazepam binding inhibitor, a natural ligand of an allosteric regulatory site of the gamma-aminobutyric acid type A receptor.

Diazepam binding inhibitor (DBI) is a protein that displaces ligands bound to the beta-carboline/benzodiazepine recognition site, an allosteric modulatory site of the type A gamma-aminobutyric acid receptor complex. An incomplete rat cDNA clone coding for DBI was isolated. This rat sequence was utilized to identify a cDNA clone that encoded the entire 104 residues of human DBI. This sequence was engineered for expression in E. coli, and recombinant DBI exhibits identical biochemical and antigenic characteristics of natural human DBI. DBI is encoded by a multigene family of at least five members, but a single gene appears to account for the majority of DBI expression. DBI is expressed in a tissue-specific manner. Expression is found in central nervous system tissues and appears to extend to peripheral tissues rich in the peripheral type of high-affinity benzodiazepine recognition sites. The role of these sites and DBI in adrenal gland, testis, and kidney remains to be determined.

Amino Acid Sequence↗
PubMed1986

Study of an octadecaneuropeptide derived from diazepam binding inhibitor (DBI): biological activity and presence in rat brain.

An endogenous brain neuropeptide with 104 amino acid residues that modulates gamma-aminobutyric acid receptor function was termed DBI because it displaces diazepam from its specific brain binding sites. Tryptic digestion of DBI generates an octadecaneuropeptide (ODN) that is more potent than the parent compound in the displacement of specifically bound beta-[3H]carboline-3-carboxylate methyl ester [( 3H]BCCM) and in proconflict action (Vogel test in thirsty rats). The proconflict action of ODN is antagonized by the imidobenzodiazepinone Ro 15-1788, which is a specific antagonist of beta-carboline and benzodiazepine recognition sites. The ODN amino acid sequence is Gln-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. The pharmacological properties associated with this sequence were confirmed by comparing the activity of ODN generated from tryptic digestion of DBI with that of ODN obtained by synthesis. Amidation of the terminal lysine of ODN produces a peptide (ODN-NH2) devoid of pharmacological activity. Three peptides containing the COOH-terminal segment of ODN were synthesized. All these peptides [Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys (octapeptide), Pro-Gly-Leu-Leu-Asp-Leu-Lys (heptapeptide), and Gly-Leu-Leu-Asp-Leu-Lys (hexapeptide)] express the displacing and proconflict actions of ODN. In primary cultures of cerebellar granule cells of rat, DBI, ODN, octapeptide, heptapeptide, and hexapeptide preferentially displace [3H]BCCM over [3H]flunitrazepam; moreover, they displace bound [3H]BCCM completely but [3H]flunitrazepam only by 50%. These data suggest that ODN includes a specific ligand for the gamma-aminobutyric acid receptor regulatory site occupied by beta-carbolines. Using rabbit antibodies directed against the NH2-terminal portion of ODN, we detected ODN-like material in rat brain homogenates. However, whether this material is identical to the ODN generated by tryptic digestion of DBI remains to be established.

Amino Acid Sequence↗
PubMed1986

Variations of supraorbital bony structures in Sienese skulls.

Various non-metrical traits were examined in the supraorbital region in a series of skulls of recorded sex (147 males, 130 females), age (18-80 years) and provenance (Siena and surroundings), using a standard method of categorization and notation introduced by two of the authors in 1983. The results are discussed from a topographic-anatomical point of view in relation to trait variation and with respect to the usefulness of the method applied for population studies.

Adult↗