ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb13381.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The regional distribution of inositol 1,4,5‐trisphosphate (InsP3), inositol 1,3,4,5‐tetrakisphosphate (InsP4), and ryanodine binding sites has been characterised and compared in the rat brain using radioligand binding assays. Cortical [3H]InsP3binding indicated similar positional and stereospecificity as observed in other tissues, with 100‐fold selectivity for lnsP3over InsP4. Similarly, high‐affinity [32P]InsP4binding also showed a high degree of positional specificity, with a 1,000‐fold selectivity for InsP4over InsP3. Initial characterisation of [3H]ryanodine binding to cortical membranes demonstrated that specific binding was highly dependent on high salt and micromolar Ca2+concentrations and inhibited by Ca2+levels of>1 mM. [3H]‐Ryanodine binding was also enhanced by β,γ‐methylene‐adenosine 5′‐trisphosphate and caffeine and inhibited by magnesium and ruthenium red (Ki= 0.81 μM). However, dantrolene (300 μM) was ineffective on the binding. Therefore, although the results indicate a greater similarity to the binding properties of the Ca2+‐induced Ca2+release channel isoform present in skeletal, rather than cardiac, muscle, it does not appear to be identical. Detailed binding analysis of ryanodine and polyphosphate sites, with the exception of ruthenium red, indicated no interaction between binding sites. Ruthenium red markedly enhanced the binding of both [3H]InsP3and [32P]InsP4, an effect most probably due to nonspecific complex formation. Regional binding of InP3, InsP4, and ryanodine in the rat brain was of similar affinity for each ligand in each area, but the density profile for each ligand was clearly different. The highest density of InsP3sites was in the cerebellum, whereas the highest density of ryanodine sites was in the hippocampus. High‐affinity InsP4sites showed less regional diversity, with highest densities in the cerebellum, cortex, and hippocampus. However, in each area studied the density of sites followed the

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb13382.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Cultured astrocytes have been shown to secrete various neuropeptides and the neuropeptide processing enzyme, carboxypeptidase E (CPE). The secretion of CPE enzymatic activity from astrocytes has been shown previously to be increased approximately twofold by treatment with tetradecanoylphorbol 13‐acetate (TPA), a phorbol ester. In this study, metabolic labeling with [35S]Met was utilized to examine the effect of TPA on the biosynthesis of CPE protein in cultured astrocytes and in AtT‐20 cells, a pituitary‐derived cell line. Treatment of astrocytes with 0.1 μg/ml TPA for 24 h caused an 80% increase in the level of radiolabeled CPE in both the media and the cells, indicating that the synthesis of CPE was stimulated by the TPA. AtT‐20 cells also secreted more radiolabeled CPE in response to TPA, but this increase was offset by a proportional decrease in the cellular level of radiolabeled CPE, and synthesis of CPE was not stimulated in this cell line. Northern blot analysis demonstrated that 0.1 μg/ml TPA elevated CPE mRNA by approximately 50% in cultured astrocytes but not in AtT‐20 cells. Quantitative in situ hybridization studies demonstrated that the TPA‐induced increase in CPE mRNA expression was largely due to increases in the number of cells expressing CPE mRNA, although for astrocytes from some brain regions the average level of CPE mRNA per cell was also elevated by TPA. These results suggest that astrocytes can be induced to express CPE, which is consistent with a role for astrocytes in intercellu

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb13383.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:We have shown previously that chronic hyperammonemia increases, in brain, the polymerization of microtubules that is regulated mainly by the level and state of phosphorylation of microtubule‐associated protein 2 (MAP‐2). Activation of theN‐methyl‐d‐aspartate (NMDA) receptor dephosphorylates MAP‐2. Because we have found that acute ammonia toxicity is mediated by the NMDA receptor, we have tested the effect of high ammonia levels on MAP‐2 in brain. Microtubules isolated from rats injected intraperitoneally with 6 mmol/kg ammonium acetate showed a marked decrease of MAP‐2. Also, the amount of MAP‐2 in brain homogenates, determined by immunoblotting. was markedly reduced, presumably by proteolysis. The content of MAP‐2 was decreased by ∼75% 1‐2 h after ammonium injection and returned to normal values after 4 h. Proteolysis of MAP‐2 was prevented completely by injection of 2 mg/kg MK‐801, a specific antagonist of the NMDA receptor, suggesting that proteolysis is mediated by activation of this receptor.l‐Carnitine, which protects rats against ammonia toxicity, also prevented MAP‐2 degradation. Because activation of the NMDA receptor increases [Ca2+]i, we determined whether rat brain contains a Ca2+‐dependent protease that selectively degrades MAP‐2. We show that there is a cytosolic Ca2+‐dependent protease that degrades MAP‐2, but no other brain proteins. The protease has been identified tentatively as calpain I, for it is inhibited by a specific inhibitor of this protease. Our results suggest that ammonium injection activates the NMDA receptor, leading to an increase in [Ca2+]i, which activates calpain I. This, in turn, selectively degrades MAP‐2. Possible implications in chronic hyperammonemic states and in the me

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb13384.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:N‐Acetylaspartylglutamate (NAAG) is the most abundant neuropeptide in the mammalian nervous system. Considerable data support the hypothesis that NAAG is synaptically released in a manner consistent with neurotransmission. Primary murine brain cultures containing neurons and glia expressed 1.2‐3.5 nmol of NAAG/mg of protein. In contrast to conclusions drawn from immunohistochemistry, pure glial cultures also expressed high levels of NAAG (0.6‐2.11 nmol/mg of protein). These data suggest that although a subpopulation of neurons contains very high NAAG levels, micromolar concentrations of the peptide also are present in glia. Both culture types demonstrated robust extracellular peptidase activity when incubated with NAAG, as well as peptide transport. Uptake of [3H]NAAG was both temperature and sodium dependent, yet relatively insensitive to the presence of extracellular glutamate. These results indicate that synaptically released NAAG, as well as that which may be released from glia, is removed from the extracellular space by direct uptake as well as the robust enzymatic degradation of the peptide. A kinetic analysis of this NAAG transport (estimatedKm= 1.8 μM) suggests a high‐affinity NAAG transport system. The balance of the two processes of direct peptide uptake and peptide hydrolysis would markedly influence the sequence of receptor‐mediated events that follow NA

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb13385.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The distribution of a novel calcium‐binding protein with a molecular mass of 18 kDa (CBP‐18) in the rat brain was studied by means of biochemical methods and immunohistochemistry on cryostat‐sectioned tissue and compared with staining patterns of parvalbumin on adjacent sections. The biochemical analysis revealed high levels of CPB‐18 in cortex and cerebellum, low levels in the lungs, and undetectable levels in all other tissues tested. Immunohistochemically, the polyclonal rabbit‐derived antibody for CPB‐18 showed selective affinity with periglomerular cells and dendrites in the olfactory bulb. Distinct immunostaining of scattered cells and their proximal dendrites was found in the anterior olfactory nuclei and in the perirhinal and entorhinal cortex. Strong staining of neuropil with recognizable but diffusely outlined cells was observed in the retrosplenial cortex, central amygdala, hippocampal rudiment, septum, area preoptica, hypothalamus, colliculus superior, and parabrachial nuclei. The cerebellum showed strong neuropil staining of both the molecular and the granule cell layer. Less intense neuropil staining and a few scattered cells were found in the neocortex, the remaining basal forebrain, and in the entire brainstem. Immunoreactivity was barely detectable or missing in the striatum, the hippocampus, the thalamus, and in the colliculus inferior. Thus, CPB‐18 shows a unique staining pattern in the CNS, different from all other Ca2+‐binding proteins

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb13386.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The technique of intracerebral microdialysis was used to assess the effect of stress on the extracellular concentrations of excitatory amino acids, glutamate and aspartate, in the rat medial prefrontal cortex, hippocampus, striatum, and nucleus accumbens. A 20‐min restraint procedure led to an increase in extracellular glutamate in all regions tested. The increase in glutamate levels was significantly higher in the prefrontal cortex than that observed in other regions. With the exception of the striatum, extracellular levels of aspartate were increased in all regions. Furthermore, the increase in aspartate levels was significantly higher in prefrontal cortex compared to hippocampus and nucleus accumbens. Local perfusion of tetrodotoxin during the restraint procedure significantly decreased the stress‐induced increase in extracellular excitatory amino acids. In order to ensure that the above results were not an artifact of restraint not associated with stress (e.g., decreased mobility), we also examined the effect of swimming stress on the extracellular levels of excitatory amino acids in selected regions, i.e., striatum and medial prefrontal cortex. Both regions displayed a significant increase in extracellular levels of aspartate and glutamate following 20 min of swimming in room temperature water. This study provides direct evidence that stress increases the neuronal release of excitatory amino acids in a regionally selective manner. The implications of the present findings for stress‐induced catecholamine release and/or hippocampal degeneration are disc

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb13387.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:To learn whether or not the levels of β‐amyloid protein precursor (APP) and τ mRNAs are related to the formation of β‐amyloid and neurofibrillary tangles, we quantified these mRNA levels in three cortical regions of 38 aged human brains, which were examined immunocyto‐chemically for β‐amyloid and tangles. Marked individual variabilities were noted in APP and τ mRNA levels among elderly individuals. The mean APP mRNA level was slightly reduced in the β‐amyloid plaque (++) group, but not in the plaque (+) group, compared to the plaque (−) group. Some brains in the plaque (−) group showed increased APP expression, the extent of which was not seen in the plaque (+)or(++) group. The differences in the mean τ mRNA levels were not statistically significant among the tangle (−), (+), and (++) groups. These results show that β‐protein and τ deposition do not accompany increased expression of the APP and τ genes, respectively, and thus suggest that factors other than gene expression may be at work in the progression of β‐amyloid and/or tangle for

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb13388.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The transforming growth factors‐β (TGFs‐β) are multifunctional peptide‐growth factors that have been localized in neuronal and glial cells of the C'NS of mice. rats, and chick embryos. We tested the TGF‐β isoforms 1. 2. and 3 for their protective ctlects against neuronal degeneration caused by cytotoxic hypoxia or by the excitatory amino acid i ‐glutamate. A cytotoxic hypoxia was induced in cultured chick embryo telencephalic neurons by adding I m.l/ sodium cyanide to the culture medium tor a period of 30min. I reatment with TGF‐81 (1‐30 ng/ml) led to a statistically significant increase in cell viability, neuronal ATP levels. and protein content of the cultures assessed 72 h after the toxic insult. TGF‐33 was able to reduce the cyanide‐induced neuronal damage at concentrations of 0.3 and 1 ng/ ml. whereas TGF‐33, only showed neuroprotective activity at concentrations of 30 and 50 ng/ml. Both pre‐ and posttreatment with TGF‐31, also prevented the degeneration of cultured chick embryo telencephalic neurons that had been exposed to I mM L‐glutamate in a buffered salt solution for a period of 60 min. Furthermore, TGF‐β1 (0.3‐3 ng/ml). and to a lesser extent TGF‐β3 (0.1‐1 ng/ml). significantly reduced excitotoxic injury of cultured neurons from rat cerebral cortex that had been exposed to serum‐free culture medium supplemented with 1 m.M L‐glutamate. These results demonstrate that the TGFs‐β are able to prevent the degeneration of primary neuronal cultures, which was caused by energy depletion and activation of gluta

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb13389.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The characteristics of the serotonin (5‐HT) output in the dorsal and median raphe nuclei of the rat were studied using in vivo microdialysis. The basal output of 5‐HT increased after KC1 was added to the perfusion fluid. In contrast, neither the omission of calcium ions nor the addition of 0.5 nM tetrodotoxin affected dialysate 5‐HT or 5‐hy‐droxyindoleacetic acid (5‐H1AA). Reserpine did not decrease the output of 5‐HT and 5‐HIAA 24 h later and p‐chloroamphetamine increased 5‐HT in both vehicle‐ and reserpine‐treated rats severalfold. 8‐Hydroxy‐2‐(di‐n‐pro‐pylamino)tetralin (8‐OH‐DPAT), at 1 or 10 μM, perfused into the raphe did not change the outputs of 5‐HT or 5‐HIAA. Higher doses (0.1, Land 10 mM) increased extracellular 5‐HT in the raphe, probably via an inhibition of uptake. In animals bearing two probes (raphe nuclei and ventral hippocampus), only the 10 vaM dose of 8‐OH‐DPAT perfused into the raphe decreased the hippocampal output of 5‐HT and 5‐HIAA. The systemic injection of 0.1 mg/kg 8‐OH‐DPAT decreased dialysate 5‐HT and 5‐HIAA in the raphe and hippocampus. These results suggest that extracellular 5‐HT in raphe nuclei originates from a cytoplasmic pool and is not dependent on either nerv

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb13390.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY