摘要:

Abstract:Activation of phosphoinositide metabolism is an early event in signal transduction for a number of neurotransmitters and hormones. In primary cultures of rat neurocortical cells, various excitatory amino acids stimulate inositol phosphate production with a rank order of potency of quisqualate>ibotenate>glutamate>kainate,N‐methyl‐d‐aspartate>α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole propionate. This response to excitatory amino acids was insensitive to a variety of excitatory amino acid antagonists including 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, 3–3(2‐carboxypiperazine‐4‐yl)propyl‐l‐phosphonate, and 2‐amino‐4‐phosphonobutyrate. The individual responses of quisqualate‐, ibotenate‐, and kainate‐stimulated inositol phosphate production were not additive. These results suggest that phosphoinositide metabolism activated by excitatory amino acids is mediated by a unique quisqualate‐preferring receptor that is not antagonized by knownN‐methyl‐d‐aspartate and non‐N‐methyl‐d‐aspartate antagonists, and is relati

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01192.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:A passage of choline from blood to brain and vice versa has been demonstrated in vivo. Because of the presence of the blood‐brain barrier, such passage takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, the choline transport properties of cerebral capillary endothelial cells have been studied in vitro. Bovine endothelial cells in culture were able to incorporate [3H]choline by a carrier‐mediated mechanism. Nonlinear regression analysis of the uptake curves suggested the presence of two transport components in cells preincubated in the absence of choline. One component showed aKmof 7.59 ± 0.8 μMand a maximum capacity of 142.7 ± 9.4 pmol/2 min/mg of protein, and the other one was not saturable within the concentration range used (1–100 μM). When cells were preincubated in the presence of choline, a single saturable component was observed with aKmof 18.5 ± 0.6 μMand a maximum capacity of 452.4 ± 42 pmol/2 min/mg of protein. [3H]Choline uptake by endothelial cells was temperature dependent and was inhibited by the choline analogs hemicholinium‐3, deanol, and AF64A. The presence of ouabain or 2,4‐dinitrophenol did not affect the [3H]choline transport capacity of endothelial cells. Replacement of sodium by lithium and cell depolarization by potassium partially inhibited choline uptake. When cells had been preincubated without choline, recently transported [3H]choline was readily phosphorylated and incorporated into cytidine‐5′‐diphosphocholine and phospholipids; however, under steady‐state conditions most (63%) accumulated [3H]choline was not metabolized within 1 h. Only part of the labelled intracellular free choline was transported back into the incubation medium. Because of their special location, transport properties, and metabolism, cerebral capillary endothelial cells may provide a gating mechanism that can contribute to the physiological control of choline concentration in the b

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01193.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The physiological function in brain of glycogen and the enzyme catalyzing the rate‐limiting step in glycogenolysis, glycogen phosphorylase (EC 2.4.1.1), is unknown. As a first step toward elucidating such a function, we have purified bovine brain glycogen phosphorylase isozyme BB 1,700‐fold to a specific activity of 24 units/mg protein. When analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and subsequent silver staining, a single major protein band corresponding to an apparent molecular mass of 97 kDa was observed. Mouse monoclonal antibodies raised against the enzyme were purified and shown to be monospecific as indicated by immunoblotting. Immunocytochemical examination of astroglia‐rich primary cultures of rat brain cells revealed a colocalization of glycogen phosphorylase with the astroglial marker glial fibrillary acidic protein in many cells. The staining for the enzyme appeared at two levels of intensity. There were other cells in the culture showing no specific staining under the experimental conditions employed. Neurons in neuron‐rich primary cultures did not show positive staining. The data suggest that glycogen phosphorylase may be predominantly an astroglial enzyme and that astroglia cells play an important role in the energy metabolism of

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01194.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The effects of forskolin (1 μM) and EGTA (5 mM) on indirect cyclic AMP responses in slices of guinea‐pig cerebral cortex were examined. Forskolin had little effect on the direct 2‐chloroadenosine‐stimulated cyclic AMP response. However, it completely abolished the glutamate‐induced augmentation of this response. In contrast, forskolin had very little effect on the indirect cyclic AMP responses to noradrenaline, 5‐hydroxytryptamine, and histamine. Conversely, rapid removal of extracellular calcium with EGTA 2 min before addition of the indirectly acting agent markedly reduced the augmentation responses produced by these latter agonists, but had little effect on the glutamate augmentation. When EGTA was added once a steady level of cyclic AMP had been achieved with the indirect agents, it was without effect on any of the responses. Thus, calcium appears to have a role in the early, but not the later, stages of the noradrenaline, 5‐hydroxytryptamine, and histamine responses. A role for protein kinase C in the glutamate augmentation response was suggested, because forskolin inhibited the augmentation of the 2‐chloroadenosine response produced by phorbol esters (which mimic the actions of diacylglycerol in activating protein kinase C). We conclude that there is more than one mechanism by which the augmentation of cyclic AMP respo

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01195.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Amphetamine facilitates the release of dopamine from nerve terminals, but the mechanisms underlying this effect have not been fully delineated. The present experiments were designed to test the extent to which amphetamine‐induced dopamine release is dependent on impulse flow and autoreceptor function in dopaminergic neurons. Rats were pretreated with a low dose of apomorphine (0.05 mg/kg) to inhibit dopamine neuronal activity, and the striatal dopaminergic response to amphetamine (0.5 mg/kg) was assessed by in vivo dialysis in freely moving animals. Consistent with previous results, apomorphine alone substantially decreased, whereas amphetamine increased, striatal dialysate dopamine concentrations. However, whereas apomorphine pretreatment decreased the locomotor response to amphetamine, the amphetamine‐induced increase in dialysate dopamine was unaffected. These results indicate that amphetamine‐facilitated dopamine release is independent of neuronal firing and autoreceptor regulation, consistent with the putative accelerative exchange‐diffusion mechanism of amphetamine‐induced dopamine release. Other possible mechanisms underlying the inhibitory effects of apomorphine on amphetamine locomotor activation are

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01196.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Tritium‐labeled hemicholinium‐3 ([3H]HC‐3) was used to characterize the sodium‐dependent high‐affinity choline carrier sites in rat striatal preparations. In an earlier study, we had shown that [3H]HC‐3 labels choline carrier sites with high and low affinities and had suggested that the low‐affinity sites represent “functional” carrier sites. The objective of the present study was to examine the mechanisms involved in the regulation of the two affinity states of [3H]HC‐3 binding. Here, we demonstrate that these two affinity states are totally interconvertible; addition of 0.1 mMATP in the binding assay medium quantitatively converted all the binding sites to the low‐affinity state, whereas addition of 1 mM β,γ‐methylene 5′‐ATP quantitatively converted all the binding sites to the high‐affinity state. Preincubation of the tissue (for 15 min at 37°C) before the binding assay also converted the binding sites to the high‐affinity state, whereas supplementation of the assay medium with ATP (0.5 mM) again induced expression of the low‐affinity state of the binding sites. This effect of ATP was found to be selective for this nucleotide. Neither ADP (1 mM) nor cyclic AMP could mimic such an effect. Other nucleotide triphosphates–CTP (0.5 mM) and GTP (0.5 mM)–also could not substitute for ATP. GTP, however, caused nearly a 35% reduction in the number of binding sites, accompanying a loss of the low‐affinity component of binding. This effect of GTP was also shared by 5′‐guanylyl‐imidodiphosphate but not by GDP or cyclic GMP. This ATPdependent low‐affinity conversion of [3H]HC‐3 binding sites requires divalent metal ions. Tissue treated with EDTA did not elicit the ATP effect; however, the sensitivity to ATP could be restored fully by supplementation with Ca2+and Mg2+. Neither a Ca2+/phospholipid‐dependent protein kinase inhibitor (staurosporin) nor Ca2+/calmodulin‐dependent protein kinase inhibitors (trifluoperazine and W‐7) blocked the Ca2+‐dependent ATP‐induced low‐affinity conversion of [3H]HC‐3 binding sites. The low‐affinity conversion could, however, be blocked by gossypol, an agent shown previously to be an inhibitor of certain calcium‐dependent protein kinases. In a similar concentration range, gossypol also inhibited synaptosomal [14C]choline uptake. Staurosporin was inactive as a choline uptake inhibitor, whereas calmodulin antagonists potently inhibited choline uptake with the rank order of calmidazolium>pimozide>trifluoperazine>W‐7>W‐13>W‐5>W‐12. We suggest that the Ca2+‐dependent ATP‐mediated modification of choline carrier sites is important in the activation of the choline carrier. The sensitivity of the synaptosomal choline uptake system to calmodulin inhibitors and the relative insensitivity of [3H]HC‐3 binding parameters to these agents suggest that calmodulin‐dependent regulation of choline uptake is mediated through a site other than [3H]HC‐3 recognition sites or through a certain modif

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01197.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Induction of the 70‐kDa heat shock protein, hsp70, was evaluated in cultured cerebellar astrocytes and granule cell neurons subjected to a hyperthermic stress, using a monoclonal antibody and an oligonucleotide probe that selectively recognize stress‐inducible species of hsp70‐related proteins and RNAs, respectively. Immunoblots of cultures enriched in either granule cells or astrocytes, and immunocytochemical localization studies in cocultures of these cell types, demonstrated that hsp70 induction was restricted to the astrocyte population. Amino acid incorporation experiments showed little difference in the loss and recovery of overall protein synthesis activity in these two cell types following transient hyperthermic stress. RNA blot hybridizations confirmed the preferential glial induction of hsp70. In vivo immunocytochemical studies in brains of adult rats following hyperthermia were consistent with earlier observations that suggested a primarily glial and vascular localization of the heat shock response in most brain regions, although the intense immunoreactivity in the cerebellar granule cell layer suggests that there is induction of hsp70 in these neurons under in vivo conditions. These results suggest the potential value of such defined cell cultures in identifying mechanisms responsible for differences in the heat shock response of various cell types in vitro, and in revealing factors that may account for the apparent absence of the stress response in cultured cerebellar granule cell ne

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01198.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The intent of this work was to elucidate the mechanism by whichN‐methyl‐d‐aspartate (NMDA) receptor agonists inhibit a second messenger system, namely, the stimulation of phosphoinositide (PI) hydrolysis activated by muscarinic cholinergic receptor agonists. NMDA inhibited cholinergic stimulation of PI hydrolysis in a dose‐ and time‐dependent manner. NMDA exerts this effect indirectly through channel activation, because both MK‐801 andN‐[1‐(2‐thienyl)cyclohexyl]piperidine (TCP) prevented this action. Prevention of the NMDA effect by removal of sodium, but not calcium, from the incubation buffer suggested that depolarization may be the responsible mechanism. Depolarization alone proved sufficient to inhibit cholinergic activation of PI hydrolysis, because both veratridine and an elevated extracellular potassium level inhibited cholinergic stimulation of PI hydrolysis. The effect of NMDA appeared to require sodium flux through NMDA channels rather than through voltage‐dependent sodium channels, because tetrodotoxin failed to inhibit the effect of NMDA. In correlative electrophysiologic experiments, NMDA profoundly inhibited evoked excitatory postsynaptic potentials and population action potentials of CA1 neurons, an effect almost certainly due to depolarization. The dose and time course of the electrophysiologic effects correlated well with the biochemical effects. Taken together, the data support the assertion that NMDA receptor activation inhibits PI hydrolysis by depolarization mediated by sodium flux th

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01199.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Severe cerebral ischaemia has been repeatedly shown to provoke a massive increase in striatal extracellular dopamine (DA). These experiments were undertaken to determine the duration of the DA increase produced by transient ischaemia, and the fate of the released DA during recirculation. Experiments were performed in anaesthetised rats subjected to 20 min of cerebral ischaemia, followed by 80 min of reperfusion, before cardiac arrest. Measurements of catechols were made in the striatum using in vivo differential pulse voltammetry (DPV), each 4 min, throughout the experiment and for 60 min after cardiac arrest. DPV data were substantiated with intracerebral dialysis; 20‐min dialysate samples were analysed for DA and homovanillic acid (HVA) using HPLC. In 6 of 11 rats, ischaemia induced a massive DA release in the striatum, resulting in a marked increase in extracellular levels (350–1,200%), which persisted throughout ischaemia. DPV and intracerebral dialysis demonstrated that DA was totally cleared from the extracellular space within minutes of reperfusion, whereas both its acidic metabolites (3,4‐dihydroxyphenylacetic acid and HVA) increased slightly. These results indicate that DA released during 20‐min ischaemia is rapidly cleared during reperfusion, mainly via reuptake. In the five other rats, only a relatively small and transient increase in the DPV catechol peak was detectable, cleared before the end of ischaemia, probably reflecting less severe ischaemia; small or no changes were detectable in the corresponding dialysate. The latter data suggest that different change(s) in the nigrostriatal dopaminergic system may occur, according to the severity of is

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01200.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Aspartate (Asp) and/or glutamate (Glu) have been proposed as putative excitatory transmitters released from synaptic terminals of the olivo‐cerebellar climbing fiber afferents to the Purkinje cells. Investigations of the climbing fiber transmitter(s) separately for hemispheres and vermis were performed to examine whether the current controversy over the role of Asp as a neurotransmitter in the climbing fibers may be due to topographic differences. K+‐induced Ca2+‐dependent release of endogenous substances was investigated in slices of cerebellar hemisphere and vermis of control rats and those deprived of climbing fibers by 3‐acetylpyridine (3‐AP) treatment. A release of Asp and Glu, as well as a small but significant release of homocysteic acid (HCA) was confirmed in control rats. Climbing fiber deprivation by 3‐AP treatment reduced the stimulated release of Asp by 48% in slices of cerebellar hemispheres, but not in vermis. Climbing fiber deprivation completely abolished the release of HCA in both hemispheres and vermis. The release of HCA, Asp, and Glu from slices of control and climbing fiber‐deprived rats evoked by 50 mMK+was>90% Ca2+‐dependent. These results support the hypothesis that Asp is a transmitter candidate of the climbing fibers projecting to the cerebellar hemispheres, but not to the vermis, and provide the first evidence that HCA can be linked to a s

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01201.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY