Identification of three transcriptional regulatory elements in the rat mitochondrial benzodiazepine receptor-encoding gene.

The sequence upstream from the first exon in the rat mitochondrial benzodiazepine receptor-encoding gene (MBR) was analyzed for transcriptional promoter activity by three techniques: promoter deletion analysis in vectors containing the gene cat encoding chloramphenicol acetyltransferase, electrophoretic mobility shift analysis (EMSA) and DNase I protection assay. All three methods are in uniformity with the identification of at least three regulatory elements corresponding to locations -51/-33, -267/-249 and -555/-526. The most distal and proximal domains are positive-acting, whereas the element at -267/-249 acts in a negative manner.

Aberrant splicing of rat steroid 17 alpha-hydroxylase transcripts.

Polymerase chain reaction amplification of the cDNA encoding steroid 17 alpha-hydroxylase (P450c17) demonstrated very low levels of this transcript and a shorter variant of still lower abundance in rat adrenals and brain. Sequence analysis of the two amplified products revealed that the shorter variant resulted from a deletion of the second exon which does not maintain the open reading frame, suggesting that faulty splicing gave rise to this minor species.

Identification and purification of a 10-kilodalton protein associated with mitochondrial benzodiazepine receptors.

The isoquinoline carboxamide photoaffinity probe PK14105, a ligand with selectivity for mitochondrial benzodiazepine receptors, has been established to photolabel an 18-kDa protein. When this radioactive probe is used to photolabel rat mitochondrial preparations, a protein of 10 kDa, in addition to the 18-kDa protein, is identified following electrophoretic separation and extended autoradiography. These proteins are referred to herein as pk10 and pk18, respectively.

Comparison of repetitive elements in the third intron of human and rodent mitochondrial benzodiazepine receptor-encoding genes.

The third intron of the mitochondrial benzodiazepine receptor (MBR)-encoding gene was sequenced from hamster, mouse and human. The rodent species were found to include an Alu-like sequence, as was first discovered in the rat gene. Differences with the rat intron were evident by an insertion of an additional B1 element in the hamster and the introduction of a complete and two partial B2 sequences in the mouse intron.

Okadaic acid increases nerve growth factor secretion, mRNA stability, and gene transcription in primary cultures of cortical astrocytes.

Neonatal rat cortical astrocytes in primary culture synthesize and secrete nerve growth factor (NGF) in response to cytokines, growth factors, and activators of protein kinases. To further implicate a protein phosphorylation mechanism in the regulation of NGF expression, astrocytes were treated with okadaic acid and calyculin A, inhibitors of phosphoprotein phosphatases 1 and 2A. Okadaic acid dramatically increased both NGF mRNA content (50-fold) and NGF secretion (100-fold) in astrocytes, while calyculin A, which has a spectrum of phosphatase inhibitory activity different from okadaic acid, failed to augment NGF expression.

Exposure of neuronal cultures to K+ depolarization or to N-methyl-D-aspartate increases the transcription of genes encoding the alpha 1 and alpha 5 GABAA receptor subunits.

The transcription rates of the alpha 1, alpha 5, and alpha 6 gamma-aminobutyric acidA receptor subunit genes were analyzed in cultures maintained in low KCl (12.5 mM), in low KCl treated with NMDA (10 microM), and in high KCl (25 mM). alpha 1 and alpha 5 transcription rates were significantly increased in response to NMDA or high KCl treatment, while alpha 6 and cyclophilin transcription rates were not changed by either condition.

Changes in gamma-aminobutyrate type A receptor subunit mRNAs, translation product expression, and receptor function during neuronal maturation in vitro.

The amounts of mRNAs encoding alpha 1, alpha 6, beta 2, beta 3, gamma 2, and delta subunits of gamma-aminobutyrate type A (GABAA) receptors and the gold immunolabeling density of their translation products were monitored during the growth of neonatal rat granule cells in primary culture. We investigated possible correlations (i) between temporal changes in mRNA content and expression density of their respective translation products and (ii) between the quantitative changes of receptor subunit expression, the GABA EC50 for Cl- channel activation, and diazepam efficacy in modulating GABA action on the Cl- channels. At 3 days in vitro, the amount of GABAA receptor subunit mRNAs and the expression of their respective translation products were very low.

Temporal and depolarization-induced changes in the absolute amounts of mRNAs encoding metabotropic glutamate receptors in cerebellar granule neurons in vitro.

Cerebellar granule neurons in primary culture express metabotropic glutamate receptors (mGluRs) coupled to the stimulation of phosphoinositide hydrolysis and to the inhibition of cyclic AMP (cAMP) formation. To evaluate which mGluR mRNAs are expressed in granule neurons under different depolarizing conditions, we measured the absolute amounts of selected receptor mRNAs in neurons cultured for 3-13 days in the presence of either 10 or 25 mM KCl. mGluR-specific primer pairs and internal standards, corresponding to unique regions of mGluR1a, mGluR2, mGluR3, mGluR4, and mGluR5, were constructed and used in a competitive PCR-derived assay to quantify the corresponding mRNA levels.

Transcriptional and posttranscriptional mechanisms involved in the interleukin-1, steroid, and protein kinase C regulation of nerve growth factor in cortical astrocytes.

Neonatal rat cortical astrocytes in primary culture synthesize and secrete nerve growth factor (NGF). Treatment of astrocytes with interleukin-1 beta (IL-1) or the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) increased NGF mRNA content by six- to 10-fold, followed in time by increases in cell content and cell secretion of NGF. Dexamethasone potently inhibited the effects of IL-1 and TPA on astroglial cell NGF expression.

LIGA20, a lyso derivative of ganglioside GM1, given orally after cortical thrombosis reduces infarct size and associated cognition deficit.

A bilateral photochemically induced thrombotic lesion of rat sensorimotor cortex (approximately 3 mm in diameter and 25 mm3 in volume) is associated with a persistent cognition (learning and memory) deficit, which was evaluated with water maze tasks. The N-dichloroacetylsphingosine derivative of lysoGM1 (LIGA20) administered after the lesion either i.v.

Mitochondrial diazepam-binding inhibitor receptor complex agonists antagonize dizocilpine amnesia: putative role for allopregnanolone.

In rats trained to retain a passive avoidance response or to retrieve a learned task in the radial and water maze tests, a pretreatment with 2-hexyl-3-indoleacetamide (FGIN-1-27) (IC50 57 mumol/kg p.o.) or 4' chlorodiazepam (4'CD) (15 mumol/kg i.

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).

Studies on the phosphorylation of the 18 kDa mitochondrial benzodiazepine receptor protein.

Steroid biosynthesis activated by pituitary tropic hormones is known to be acutely regulated by cAMP acting via Protein kinase A. Because the mitochondrial benzodiazepine receptor (MBR) has been suggested to play a role in the activation of steroidogenesis, the present study investigates whether various protein kinases phosphorylate MBR. In rat and bovine adrenal mitochondrial preparations Protein kinase A, but not other purified protein kinases, was found to phosphorylate the 18 kDa MBR protein.

Acute and long-term inhibition of agonist-stimulated phosphoinositide hydrolysis by pulse treatment of cerebellar granule cells with TPA.

Acute pretreatment (30 min) of primary cultures of cerebellar granule cells with TPA (10 nM) resulted in a decrease in carbachol-and glutamate-stimulated phosphoinositide hydrolysis, but not in basal levels of PI hydrolysis. To investigate the mechanism of TPA action, phospholipase C was assayed in membranes prepared from cerebellar granule cells acutely treated with TPA. TPA had no effect on basal, GTP gamma S-, NaF-, and calcium-stimulated phospholipase C when compared with membranes prepared from vehicle-treated cells.

Intracellular sodium concentration in cultured cerebellar granule cells challenged with glutamate.

We monitored simultaneously the changes in the intracellular sodium concentration ([Na+]i) and intracellular calcium concentration ([Ca2+]i) in individual neurons from primary cultures of cerebellar granule cells loaded with sodium-binding benzofuran isophthalate and fluo-3. An application of glutamate (50 microM) in Mg(2+)-free medium containing 10 microM glycine evoked [Na+]i and [Ca2+]i increases that exceeded 60 mM and 1 microM, respectively. The kinetics of [Na+]i and [Ca2+]i decreases after the termination of the glutamate pulse were different.

Quantitative changes in alpha 1 and alpha 5 gamma-aminobutyric acid type A receptor subunit mRNAs and proteins after a single treatment of cerebellar granule neurons with N-methyl-D-aspartate.

It was previously demonstrated that daily administration of N-methyl-D-aspartate (NMDA) to primary cultures of cerebellar granule neurons for 5 days in vitro mediates an increase in the relative content of mRNAs encoding selected subunits of the gamma-aminobutyric acid (GABA)A receptor. This analysis was extended using a competitive polymerase chain reaction assay with internal standards to quantitate changes that occur in the absolute amounts of selected GABAA receptor subunit mRNAs in cerebellar granule neurons in vitro after the single administration of nontoxic doses of either NMDA or glutamate. For these studies, we focused on the alpha 1, alpha 5, and alpha 6 receptor subunit mRNAs and examined their absolute contents in cultures maintained in low KCl (12.

AMPA-selective glutamate receptor subtype immunoreactivity in the entorhinal cortex of non-demented elderly and patients with Alzheimer's disease.

The present work employed immunocytochemical techniques and examined the distribution and cytological features of the AMPA receptor subunits, GluR2/3 and GluR1 within the entorhinal cortex of non-demented elderly (NC), patients with neuropathological and clinical verification of Alzheimer's disease (AD) and patients without a clinical history of dementia yet exhibiting sufficient quantities of senile plaques to meet neuropathological criteria of Alzheimer's disease (HPND). In NC cases, GluR2/3-immunolabeled neurons were abundantly distributed throughout layers II, III, V and VI of the entorhinal cortex. In contrast, GluR1-positive cells were comparatively sparse in number and largely restricted to layers V and VI.

N-methyl-D-aspartate receptor-induced translocation of protein kinase C to the nucleus in rat cerebellar slices.

Rat cerebellar slices were incubated in absence and presence of N-methyl-D-aspartate and then used to prepare a purified nuclear fraction. The purity of the nuclear fraction was assessed by electron microscopy and measurements of Na+, K(+)-ATPase activity. The presence of protein kinase C in nuclear fractions was measured by [3H]phorbol dibutyrate binding.

Glutamate impairs neuronal calcium extrusion while reducing sodium gradient.

The rate of decrease of neuronal [Ca2+]i after an elevation induced by a glutamate pulse is much slower than that after a comparable [Ca2+]i elevation induced by a K+ depolarization. To investigate whether the [Na+]i increase taking place during the glutamate pulse reduces the rate of Ca2+ extrusion, we monitored simultaneously [Na+]i and [Ca2+]i during a K+ depolarization and a glutamate pulse lasting 1 min. The K+ depolarization evoked only a transient increase of [Na+]i from 4 mM to 13 mM, whereas the glutamate pulse increased [Na+]i to 60 mM, and this increase persisted after glutamate removal.

Persistent AMPA receptor stimulation alters [Ca2+]i homeostasis in cultures of embryonic dopaminergic neurons.

The effect of the ionotropic glutamate receptor agonist, AMPA, on intracellular Ca2+ concentrations ([Ca2+]i) was studied in dopaminergic neurons present in primary cultures of ventral tegmental mesencephalon of 14 day rat embryos. Exposure of cells to 10 microM AMPA for 1 min increased [Ca2+]i by 2-3 fold in dopaminergic and other neurons and this response was obliterated within 5 min by superfusion with AMPA-free incubation buffer. In dopaminergic neurons, 1 min or 5 min exposure to 50 microM AMPA increased [Ca2+]i 3 to 5 times over control values.

Double-immunolabelling analysis of GABAA receptor subunits in label-fracture replicas of cultured rat cerebellar granule cells.

We used double-immunolabelling with various size gold particles in label-fracture replicas to gain information on the subunit composition of GABAA receptors in clusters located in the plasma membrane of cultured cerebellar granule cells. We hypothesize that if two subunits colocalize in the same GABAA receptor, the density of the label of any given subunit decreases when another subunit of the same receptor complex is prelabelled with an antibody linked to a gold particle of larger size. Following this hypothesis, the interpretation of our results allows to suggest that a consistent receptor colocalization of beta 2/3 with alpha 1 or alpha 6 subunits may be operative in cerebellar granule cells.

Brain content of glycosphingolipids after oral administration of monosialogangliosides GM1 and LIGA20 to rats.

Natural (GM1) and semisynthetic [113-Neu-5-AcGgOse4-2-D-erythro-1,3- dihydroxy-2-dichloroacetylamide-4-trans-octadecene (LIGA20)] glycosphingolipids, given parenterally, protect neurones against glutamate-induced death without producing the side effects typical of glutamate receptor antagonists. Chronic glutamate-related neurotoxicity (e.g.

Pituitary adenylate cyclase activating polypeptide (PACAP) potently enhances tyrosine hydroxylase (TH) expression in adrenal chromaffin cells.

In primary cultured bovine adrenal chromaffin cells (BACC), pituitary adenylate cyclase activating polypeptide 1-38 (PACAP) produced a dose related increase in tyrosine hydroxylase (TH) Vmax when measured 48 hours after the beginning of the treatment; a significant increase was observed with 0.5 nM and the maximal induction of close to 2.5-fold was found with 0.