Further studies on the role of astroglia in brain neurons maturation and morphogenesis

Using in vitro cultures of dissociated brain neurons and astrocytes, we have compared the morphologies of mesencephalic and striatal neurons cultured for two days on mesencephalic and striatal astrocytes in the four possible combinations. From these comparisons, it appears that: 1. Neurons grown on co-regionalized (homotopic) astrocytes have more primary neurites and branching points than neurons grown on heterotopic astrocytes. 2. The total neuritic length is only slightly affected by the type of co-culture. 3. The branched arborization which develop faster on homotopic astrocytes present several dendritic features. Following these morphological observations, we have been able to demonstrate: 1. That mesencephalic astrocytes (but not striatal astrocytes) secrete trypsin sensitive factors different from laminin and FGF that increase the number of primary neurites and branching points but have no or little effect on total neuritic length. 2. That mesencephalic astrocytes (but not striatal astrocytes) present at their surface a 190 KD glycoprotein specifically recognized by the fucose-specific lectin UEA.     

Vasoactive Intestinal Polypeptide Receptors Linked to an Adenylate Cyclase, and Their Relationship with Biogenic Amine- and Somatostatin-Sensitive Adenylate Cyclases on Central Neuronal and Glial Cells in Primary Cultures

The presence of vasoactive intestinal polypeptide (VIP) receptors coupled to an adenylate cyclase was demonstrated on membranes of neurons or glial cells grown in primary cultures originating from the cerebral cortex, striatum, and mesencephalon of mouse embryos. A biphasic pattern of activation was observed in all these cell types, involving distinct high- and low-apparent-affinity mechanisms. The absence of additive effects of VIP and 3,4-dihydroxyphenylethylamine (DA, dopamine), isoproterenol (ISO), and 5-hydroxytryptamine (5-HT, serotonin) suggests that the peptide receptors are colocated with each of the corresponding amine receptors on neuronal membranes of the three structures studied. The nonadditivity between the VIP- and ISO-induced responses on cortical and striatal glial membranes reveals as well a colocation of VIP and beta-adrenergic-sensitive adenylate cyclases on the same cells. A subpopulation of mesencephalic glia could possess only one of the two types of receptors, as a partial additivity of the VIP and ISO responses was seen. In addition, VIP modified the characteristics of the somatostatin inhibitory effect on adenylate cyclase activity of neuronal membranes from the cerebral cortex and striatum but not from those of the mesencephalon. On striatal and mesencephalic glial membranes the somatostatin inhibitory effect was observed only in the presence of VIP. However, as previously seen with ISO, the presence of VIP did not allow the appearance of a somatostatin inhibitory response on cortical glial membranes. This suggests that cortical glia are devoid of somatostatin receptors.     

Differential involvement of cell cycle reactivation between striatal and cortical neurons in cell death induced by 3-nitropropionic acid

Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G(1) phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca(2+) and activation of calpain, a Ca(2+)-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca(2+) and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease.     

Group I metabotropic glutamate receptors mediate phospholipase D stimulation in rat cultured astrocytes

We have studied the activation of phospholipase D (PLD) by glutamate in rat cultured astrocytes by measuring the PLD-catalyzed formation of [32P]phosphatidylbutanol in [32P]Pi-prelabeled cells, stimulated in the presence of butanol. Glutamate elicited the activation of PLD in cortical astrocytes but not in cortical neurons, whereas similar glutamate activation of phosphoinositide phospholipase C was found in both astrocytes and neurons. The extent of PLD stimulation by glutamate was similar in astrocytes from brain cortex and hippocampus, but no effect was found in cerebellar astrocytes. In cortical astrocytes, the glutamate response was insensitive to antagonists of ionotropic glutamate receptors and was reproduced by agonists of metabotropic glutamate receptors (mGluRs) with a rank order of agonist potency similar to that reported for group I mGluR-mediated phosphoinositide phospholipase activation [quisqualate > (S)-3,5-dihydroxyphenylglycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid]. The response to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid was inhibited by the mGluR antagonist (S)-alpha-methyl-4-carboxyphenylglycine and, less potently, by 1-aminoindan-1,5-dicarboxylic acid and 4-carboxyphenylglycine, two antagonists of group I mGluRs that display higher potency on mGluR1 than on mGluR5. The mGluR5-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine also activated PLD in astrocytes. These findings indicate the involvement of group I mGluRs, most likely mGluR5, in the glutamate activation of PLD in cultured rat cortical astrocytes.     

Regional Characteristics of Histamine Uptake into Neonatal Rat Astrocytes

Histaminergic signalling constitutes an attractive target for treatment of neuropsychiatric disorders. One obstacle to developing new pharmacological options has been failure to identify putative specific histamine transporter responsible for histamine clearance. Although high-affinity histamine uptake was detected in neonatal cortical astrocytes, its existence in other brain regions remains largely unexplored. We investigated whether cerebellar and striatal astrocytes participate in histamine clearance and evaluated the role of organic cation transporters (OCT) in astroglial histamine transport. Kinetic and pharmacological characteristics of histamine transport were determined in cultured astrocytes derived from neonatal rat cerebellum, striatum and cerebral cortex. As well as astrocytes of cortical origin, cultured striatal and cerebellar astrocytes displayed temperature-sensitive, high-affinity histamine uptake. Exposure to ouabain or Na⁺-free medium, supplemented with choline chloride markedly depressed histamine transport in cortical astrocytes. Conversely, histamine uptake in striatal and cortical astrocytes was ouabain-resistant and was only partially diminished during incubation in the absence of Na⁺. Also, histamine uptake remained unaltered upon exposure to OCT inhibitor corticosterone, although OCTs were expressed in cultured astrocytes. Finally, histamine transport in cerebellar and striatal astrocytes was not sensitive to antidepressants. Despite common characteristics, cerebellar astrocytes had lower affinity, but markedly higher transport capacity for histamine compared to striatal astrocytes. Collectively, we provide evidence to suggest that cerebellar, striatal as well as cortical astrocytes possess saturable histamine uptake systems, which are not operated by OCTs. In addition, our data indicate that Na⁺-independent histamine carrier predominates in cerebellar and striatal astrocytes, whereas Na⁺-dependent transporter underlies histamine uptake in cortical astrocytes. Our findings implicate a role for histamine transporters in regulation of extracellular histamine concentration in cerebellum and striatum. Inhibition of histamine uptake might represent a viable option to modulate histaminergic neurotransmission.     

Neuronal lineages in chimeric mouse forebrain are segregated between compartments and in the rostrocaudal and radial planes

On the basis of neuronal phenotypes and the mode of development of the mammalian forebrain, the cerebral cortex can be subdivided into deep versus superficial layers, and the striatum into patch versus matrix compartments. Interspecific chimeric Mus musculus----Mus caroli mice were used to determine the contribution of lineage to cellular position within these forebrain compartments. Statistical analysis revealed evidence of both spatial and compartmental lineage segregation. A significant difference in genotype ratio depending on chimeric specimen was observed between areas (regardless of compartment) that were separated by greater than 300 microns in the rostrocaudal plane. Differences were observed between early-born (striatal patch and deep cortex) versus late-born (striatal matrix and superficial cortex) neurons, but not between neurons of cortex as a whole versus neurons of striatum as a whole. The difference between early- and late-born neurons was primarily due to the difference between deep and superficial cortical neurons. On a finer scale of analysis, differences in genotype ratios were seen between radially aligned deep versus superficial cortical compartments, in both the neuronal and glial populations. This evidence is consistent with an early positional and compartmental segregation of forebrain progenitor cells.     

Cortical astrocytes activated by basic fibroblast growth factor secrete molecules that stimulate differentiation of mesencephalic dopaminergic neurons.

In reactive gliosis, astrocytes undergo morphological and biochemical changes which can be mimicked in vitro by treatment with bFGF (basic fibroblast growth factor) or cAMP. To investigate the influence of activated cortical astrocytes on central nervous system (CNSD) neurons, we studied the effect of the supernatant from bFGF-treated astrocytes on the development of dopaminergic neurons from rat mesencephalon. Conditioned medium of untreated astrocytes stimulated dopamine uptake of mesencephalic cultures. After activation of astrocytes with bFGF this effect was greatly enhanced. It was significantly more potent than stimulating effects of other neurotrophic factors. The supernatant of these astrocytes increased the biochemical differentiation but not the survival of dopaminergic neurons in our cell culture system. Trypsin digestion and gel chromatography revealed that the activity was due to one or several proteins with molecular mass above 5 kDa. We excluded the participation of several factors known to be produced by astrocytes or that are neurotrophic for substantia nigra cultures. In particular, we provide evidence that bFGF, BDNF, NT-3, Il-1, Il-6, S100 beta and alpha 2-macroglobulin were not involved in the effect of the conditioned medium. In vitro stimulation of astrocytes therefore triggers the expression of currently uncharacterized factors which influence the biochemical differentiation of mesencephalic dopaminergic neurons, the cells that degenerate in Parkinson's disease.     

Metabolism and Release of Glutamate in Cerebellar Granule Cells Cocultured with Astrocytes from Cerebellum or Cerebral Cortex

Cerebellar granule cells were cocultured with astrocytes from either cerebral cortex or cerebellum in two different systems. In one system the cells were plated next to each other only sharing the culture medium (separated cocultures) and in the other system the granule cells were plated on top of a preformed layer of astrocytes (sandwich cocultures). Using astrocytes from cerebellum, granule cells developed morphologically and functionally showing a characteristic high activity of the glutamate synthesizing enzyme aspartate aminotransferase (AAT) as well as a high stimulus-coupled transmitter release regardless of the culture system, i.e., granule cells could grow on top of cerebellar astrocytes as well as next to these cells. In the case of cerebral cortex astrocytes it was found that cerebellar granule cells did not develop (11% survival) when seeded on top of these astrocytes. This was indicated by the morphological appearance of the cultures as well as by a negligible difference between the AAT activity in sandwich cocultures and astrocytes cultured alone. On the other hand, granule cells in separated cocultures with cerebral cortex astrocytes exhibited a normal morphology and a high activity of AAT as well as a large stimulus-coupled transmitter release. Cerebellar and cortical astrocytes expressed the astrocyte specific enzyme glutamine synthetase in a glucocorticoid-inducible form regardless of the culture system. The results show that under conditions of direct contact between granule cells and astrocytes, regional specificity exists with regard to neuron-glia contacts. This specificity does not seem to involve soluble factors present in the culture medium because in separated cocultures the cerebellar granule cells developed normally regardless of the regional origin of the astrocytes.     

Marked Regional Heterogeniety of 125I-Bolton Hunter Substance P Binding and Substance P-Induced Activation of Phospholipase C in Astrocyte Cultures from the Embryonic or Newborn Rat

The specific binding of 125I-Bolton Hunter substance P (125I-BHSP) was estimated on 4- to 5-week-old primary cultures of astrocytes from several brain structures and the spinal cord of 16-day-old embryonic or newborn rats. In both cases, high levels of binding of 125I-BHSP were found on intact astrocytes from the brainstem, but this binding was low or negligible on cells from the cerebral cortex, striatum, hypothalamus, and mesencephalon. In addition, hippocampal astrocytes from newborn rats were also devoid of 125I-BHSP binding sites, while a binding of 125I-BHSP (half that of brainstem cells) was observed on astrocytes from the cerebellum and spinal cord. It was also shown that this regional heterogeneity in 125I-BHSP binding was not linked to differences in the inactivation of the ligand, cell plating density. or eventual cell contaminants. Five-day-old cultures from 16-day-old embryos were used to estimate 125I-BHSP binding on neuron-enriched cultures. Specific 125I-BHSP binding was found on cells from the brainstem, mesencephalon, and hypothalamus, but neurons from the cerebral cortex or the striatum contained low or negligible amounts of 125I-BHSP binding sites. Competition studies using tachykinins and SP analogues indicated that 125I-BHSP binding sites on brainstem astrocytes (16-day-old embryos) have the pharmacological profile expected for NK1 binding sites. SP (1 microM) stimulated phosphoinositide breakdown in cells rich in 125I-BHSP binding sites (brainstem) but not in those devoid of 125I-BHSP binding (striatum).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cortical modulation of cholinergic neurons in the striatum

Previous reports suggest the existence of a cortico-striatal pathway which might use glutamate as the transmitter. In the present study, the possible influence of this pathway on striatal cholinergic neurons was investigated. Two weeks following surgical destruction of the cerebral cortex, the high affinity uptake of glutamate and choline into striatal synaptosomes was significantly reduced whereas GABA uptake was unaffected. In acute experiments (1 hour following decortication), only choline uptake was significantly reduced while the uptake of glutamate and GABA were not altered. Acute injection (2 minutes) of kainic acid into the striatum, 1 hour after decortication, reversed the effect of the decortication on choline uptake, perhaps by simulating an excitatory input to the striatum which was presumably removed by the cortical ablation. These observations are consistent with the existence of a cortical input (perhaps glutamatergic) to the striatum and suggest that striatal cholinergic neurons can be influenced by this cortico-striatal pathway.     

Brain oscillations,medium spiny neurons,and dopamine

1. The striatum is part of a multisynaptic loop involved in translating higher order cognitive activity into action. The main striatal computational unit is the medium spiny neuron, which integrates inputs arriving from widely distributed cortical neurons and provides the sole striatal output.2. The membrane potential of medium spiny neurons' displays shifts between a very negative resting state (down state) and depolarizing plateaus (up states) which are driven by the excitatory cortical inputs.3. Because striatal spiny neurons fire action potentials only during the up state, these plateau depolarizations are perceived as enabling events that allow information processing through cerebral cortex – basal ganglia circuits. In vivo intracellular recording techniques allow to investigate simultaneously the subthreshold behavior of the medium spiny neuron membrane potential (which is a reading of distributed patterns of cortical activity) and medium spiny neuron firing (which is an index of striatal output).4. Recent studies combining intracellular recordings of striatal neurons with field potential recordings of the cerebral cortex illustrate how the analysis of the input–output transformations performed by medium spiny neurons may help to unveil some aspects of information processing in cerebral cortex – basal ganglia circuits, and to understand the origin of the clinical manifestations of Parkinson's disease and other neurologic and neuropsychiatric disorders that result from alterations in dopamine-dependent information processing in the cerebral cortex – basal ganglia circuits.     

Evidence that glutaric acid reduces glutamate uptake by cerebral cortex of infant rats

The role of excitotoxicity in the cerebral damage of glutaryl-CoA dehydrogenase deficiency (GDD) is under intense debate. We therefore investigated the in vitro effect of glutaric (GA) and 3-hydroxyglutaric (3-OHGA) acids, which accumulate in GDD, on [(3)H]glutamate uptake by slices and synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Glutamate uptake was significantly decreased by high concentrations of GA in cortical slices of 7-day-old rats, but not in cerebral cortex from 15- and 30-day-old rats and in striatum from all studied ages. Furthermore, this effect was not due to cellular death and was prevented by N-acetylcysteine preadministration, suggesting the involvement of oxidative damage. In contrast, glutamate uptake by brain slices was not affected by 3-OHGA exposure. Immunoblot analysis revealed that GLAST transporters were more abundant in the cerebral cortex compared to the striatum of 7-day-old rats. Moreover, the simultaneous addition of GA and dihydrokainate (DHK), a specific inhibitor of GLT1, resulted in a significantly higher inhibition of [(3)H]glutamate uptake by cortical slices of 7-day-old rats than that induced by the sole presence of DHK. We also observed that both GA and 3-OHGA exposure did not alter the incorporation of glutamate into synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Finally, GA in vivo administration did not alter glutamate uptake into cortical slices from 7-day-old rats. Our findings may explain at least in part why cortical neurons are more vulnerable to damage at birth as evidenced by the frontotemporal cortical atrophy observed in newborns affected by GDD.     

No Role for Phospholipase A2 and Protein Kinase C in the Potentiation by α-Adrenoceptors of β-Adrenoceptor-Mediated Cyclic AMP Formation in Rat Brain

This study was undertaken to examine the role of phospholipase A2 and protein kinase C in the potentiation of beta-adrenoceptor-mediated cyclic AMP formation by alpha-adrenoceptors in rat cerebral cortical slices. Inhibition of arachidonic acid metabolism by a range of cyclooxygenase and lipoxygenase inhibitors had no effect on the potentiation of isoprenaline-stimulated cyclic AMP. Conversely, stimulation of leukotriene formation had no effect on the response to isoprenaline. The phospholipase A2 activator, melittin, stimulated cyclic AMP and potentiated the effect of isoprenaline, but these responses were not influenced by cyclooxygenase or lipoxygenase inhibitors. Indomethacin was also ineffective against the potentiation of vasoactive intestinal peptide-stimulated cyclic AMP by noradrenaline. Phorbol ester potentiated the cyclic AMP response to isoprenaline, and this potentiation was antagonized by three different putative protein kinase C inhibitors. However, the same inhibitors did not affect the alpha-adrenoceptor-stimulated enhancement of the response to isoprenaline. We have found no evidence, therefore, to support the suggestion that arachidonic acid and its metabolites and/or protein kinase C mediate the alpha-adrenoceptor modulation of beta-adrenoceptor function.     

Temporally and spatially regulated expression of a candidate G-protein-coupled receptor during cerebral cortical development

Genes expressed in layer-specific patterns in the mammalian cerebral cortex may play a role in specifying the identity of different cortical layers. Using PCR-differential display, we identified a cDNA that encodes rCNL3, a gene cloned previously by sequence homology to G-protein-coupled receptors. rCNL3 is expressed predominantly in layers 2-4 of the young rat cortex and in the developing and adult striatum. Cortical expression of rCNL3 begins postnatally at P3 and continues at high levels until around P15, while striatal expression begins at E20 and continues through adulthood. rCNL3 expression is not detectable in the ventricular zone precursors that generate the neurons of layers 2-4. The expression pattern of rCNL3 in the developing cortex suggests that rCNL3 is not involved in the initial specification of laminar fate, but rather may be involved with later differentiation events within the superficial cortical layers.     

Striatal Astrocytes Act as a Reservoir for L-DOPA

L-DOPA is therapeutically efficacious in patients with Parkinson’s disease (PD), although dopamine (DA) neurons are severely degenerated. Since cortical astrocytes express neutral amino acid transporter (LAT) and DA transporter (DAT), the uptake and metabolism of L-DOPA and DA in striatal astrocytes may influence their availability in the dopaminergic system of PD. To assess possible L-DOPA- and DA-uptake and metabolic properties of striatal astrocytes, we examined the expression of L-DOPA, DA and DAT in striatal astrocytes of hemi-parkinsonian model rats after repeated L-DOPA administration, and measured the contents of L-DOPA, DA and their metabolite in primary cultured striatal astrocytes after L-DOPA/DA treatment. Repeated injections of L-DOPA induced apparent L-DOPA- and DA-immunoreactivities and marked expression of DAT in reactive astrocytes on the lesioned side of the striatum in hemi-parkinsonian rats. Exposure to DA for 4h significantly increased the levels of DA and its metabolite DOPAC in cultured striatal astrocytes. L-DOPA was also markedly increased in cultured striatal astrocytes after 4-h L-DOPA exposure, but DA was not detected 4 or 8h after L-DOPA treatment, despite the expression of aromatic amino acid decarboxylase in astrocytes. Furthermore, the intracellular level of L-DOPA in cultured striatal astrocytes decreased rapidly after removal of extracellular L-DOPA. The results suggest that DA uptaken into striatal astrocytes is rapidly metabolized and that striatal astrocytes act as a reservoir of L-DOPA that govern the uptake or release of L-DOPA depending on extracellular L-DOPA concentration, but are less capable of converting L-DOPA to DA.     

[3H]8-Hydroxy-2-(Di-n-Propylamino)Tetralin Binding to Pre- and Postsynaptic 5-Hydroxytryptamine Sites in Various Regions of the Rat Brain

The specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]8-OH-DPAT) to 5-hydroxytryptamine (5-HT)-related sites was investigated in several regions of the rat brain. Marked differences were observed in the characteristics of binding to membranes from hippocampus, striatum, and cerebral cortex. Hippocampal sites exhibited the highest affinity (KD approximately 2 nM) followed by the cerebral cortex (KD approximately 6 nM) and the striatum (KD approximately 10 nM). Ascorbic acid inhibited specific [3H]8-OH-DPAT binding in all three regions but millimolar concentrations of Ca2+, Mg2+, and Mn2+ enhanced specific binding to hippocampal membranes, whereas only Mn2+ increased it in the cerebral cortex and all three cations inhibited specific binding to striatal membranes. Guanine nucleotides (0.1 mM GDP, GTP) inhibited binding to hippocampal and cortical membranes only. As intracerebral 5,7-dihydroxytryptamine markedly decreased [3H]8-OH-DPAT binding sites in the striatum, but not in the hippocampus, the striatal sites appear to be on serotoninergic afferent fibers. In contrast, in the hippocampus the sites appear to be on postsynaptic 5-HT target cells, as local injection of kainic acid decreased their density. Both types of sites appear to be present in the cerebral cortex. The postsynaptic hippocampal [3H]8-OH-DPAT binding sites are probably identical to the 5-HT1A subsites, but the relationship between the presynaptic binding sites and the presynaptic autoreceptors controlling 5-HT release deserves further investigation.     

Alpha 1-adrenergic receptor-mediated activation of phospholipase D in rat cerebral cortex.

We have investigated phospholipase D activity in rat brain cortical slices prelabeled with [32P]orthophosphoric acid. In the presence of ethanol (170 mM), norepinephrine stimulated, in a dose-dependent manner (EC50 = 2.2 microM), the accumulation of [32P]phosphatidylethanol as a result of phospholipase D activity. Norepinephrine-stimulated phospholipase D activity was completely inhibited by prazosin, a specific alpha 1-adrenergic antagonist (Ki = 2.8 nM). However, no accumulation of phosphatidylethanol was observed in the presence of the muscarinic agonist carbachol. The Ca2+ ionophore ionomycin and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also stimulated [32P]phosphatidylethanol accumulation in cortical slices, in a dose- and time-dependent manner, whereas the inactive phorbol, 4 alpha-phorbol 12,13-didecanoate, did not stimulate phospholipase D activity. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, two potent inhibitors of protein kinase C, inhibited PMA and ionomycin stimulation of phospholipase D activity, but did not affect the response to norepinephrine. Furthermore, the effects of PMA and norepinephrine were additive. Differences between PMA and norepinephrine stimulation of phospholipase D activity were also found with regard to the extracellular Ca2+ requirement and time course of phosphatidylethanol accumulation. No stimulation of phospholipase D activity by norepinephrine was observed in slices from cerebellum, a brain area with a low density of alpha 1-adrenergic receptors, while the effect of PMA was greater in the cerebellum than in cortical or hippocampal slices. These results strongly suggest that activation of phospholipase D in cortical slices by norepinephrine and PMA involve different mechanisms.     

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.     

Endothelin Stimulates Phospholipase D in Striatal Astrocytes

Abstract: In primary cultures of mouse striatal astrocytes prelabeled with [³H]myristic acid, endothelin (ET)-1 induced a time-dependent formation of [³H]phosphatidic acid and [³H]diacylglycerol. In the presence of ethanol, a production of [³H]phosphatidylethanol was observed, indicating the activation of a phospholipase D (PLD). ET-1 and ET-3 were equipotent in stimulating PLD activity (EC₅₀ = 2–5 n M ). Pretreatment of the cells with pertussis toxin partially abolished the effect of ET-1, indicating the involvement of a G_i/G_o protein. Inhibition of protein kinase C by Ro 31-8220 or down-regulation of the kinase by a long-time treatment with phorbol 12-myristate 13-acetate (PMA) totally abolished the ET-1-induced stimulation of PLD. In contrast, a cyclic AMP-dependent process is not involved in the activation of PLD, because the ET-1-evoked formation of [³H]phosphatidylethanol was not affected when cells were coincubated with either isoproterenol, 8-bromo-cyclic AMP, or forskolin. Acute treatment with PMA also stimulated PLD through a protein kinase C-dependent process. However, the ET-1 and PMA responses were additive. Furthermore, the ET-1-evoked response, contrary to that of PMA, totally depended on the presence of extracellular calcium. These results suggest that at least two distinct mechanisms are involved in the control of PLD activity in striatal astrocytes. Finally, ET-1, ET-3, and PMA also stimulated PLD in astrocytes from the mesencephalon, the cerebral cortex, and the hippocampus.