ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb13275.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:The activation of kainic acid and quisqualic acid receptors in cultured cerebellar granule cells stimulated the release of preaccumulatedd‐[3H]aspartate. The effect of kainate could be distinguished from that of quisqualate by its sensitivity to the antagonists kynurenic acid and 2,3‐cis‐piperidine dicarboxylic acid. At a concentration of kainic acid (50 μM) close to its half‐maximal releasing effect, simultaneous addition of quisqualic acid (10–50 μM) resulted in a significant dose‐dependent inhibition of the kainate‐induced component ofd‐[3H]aspartate release, which was monitored by the progressive decrease in sensitivity of the evoked release to kynurenic acid. In contrast, when kainic acid was used at a subeffective concentration (10 μM), addition of low doses of quisqualate (2–5 μM) resulted in a synergistic effect ond‐[3H]aspartate release. Under these conditions, the effect of the two agonists was sensitive to kynurenic acid. Kainic acid (50–100 μM) also caused a dose‐dependent, kynurenic acid‐sensitive accumulation of cyclic GMP (cGMP) in granule cell cultures. Quisqualic acid was, by itself, ineffective and prevented, in a dose‐dependent manner, the kainate‐induced cGMP formation (IC50= 5 μM). Finally, the guanylate cyclase activator sodium nitroprusside greatly enhanced cGMP formation but had no effect ond‐[3H]aspartate release. Together, these results demonstrate the existence of complex interactions between quisqualic and kainic acids and indicate that the effects of the two glutamate agonists ond‐[3H]aspartate release a

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10891.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:To determine changes in the degree of phosphorylation of the protein kinase C substrate B‐50 in vivo, a quantitative immunoprecipitation assay for B‐50 (GAP43, F1, pp46) was developed. B‐50 was phosphorylated in intact hippocampal slices with32Pior in synaptosomal plasma membranes with [γ‐32P]ATP. Phosphorylated B‐50 was immunoprecipitated from slice homogenates or synaptosomal plasma membranes using polyclonal anti–B‐50 antiserum. Proteins in the immunoprecipitate were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and the incorporation of32P into B‐50 was quantified by densitometric scanning of the autoradiogram. Only a single 48‐kilodalton phosphoband was detectable in the immunoprecipitate, but this band was absent when preimmune serum was used. The B‐50 immunoprecipitation assay was quantitative under the following condition chosen, as (1) recovery of purified32P‐labelled B‐50 added to slice homogenates or synaptosomal plasma membranes was>95%; and (2) modulation of B‐50 phosphorylation in synaptosomal plasma membranes with adrenocorticotrophic hormone, polymyxin B, or purified protein kinase C in the presence of phorbol diester resulted in EC50values identical to those obtained without immunoprecipitation. With this immunoprecipitation assay we found that treatment of hippocampal slices with 4β‐phorbol 12,13‐dibutyrate stimulated B‐50 phosphorylation, whereas 4α‐phorbol 12,13‐didecanoate was inactive. Thus, we conclude that the B‐50 immunoprecipitation assay is suitable to monitor changes in B‐

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10892.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Recent studies have demonstrated that phorbol diesters enhance the release of various neurotransmitters. It is generally accepted that activation of protein kinase C (PKC) is the mechanism by which phorbol diesters act on neurotransmitter release. The action of PKC in neurotransmitter release is very likely mediated by phosphorylation of substrate proteins localized in the presynaptic nerve terminal. An important presynaptic substrate of PKC is B‐50. To investigate whether B‐50 mediates the actions of PKC in neurotransmitter release, we have studied B‐50 phosphorylation in intact rat hippocampal slices under conditions that stimulate or inhibit PKC and neurotransmitter release. The slices were labelled with [32P]orthophosphate. After treatment, the slices were homogenized, B‐50 was immunoprecipitated from the slice homogenate, and the incorporation of32P into B‐50 was determined. Chemical depolarization (30 μMK+) and the presence of phorbol diesters, conditions that stimulate neurotransmitter release, separately and in combination, also enhance B‐50 phosphorylation. Polymyxin B, an inhibitor of PKC and neurotransmitter release, decreases concentration dependently the depolarization‐induced stimulation of B‐50 phosphorylation. The effects of depolarization are not detectable at low extracellular Ca2+concentrations. It is concluded that in rat hippocampal slices B‐50 may mediate the action of PKC in neurot

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10893.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:[3H]Kainate binding to membrane homogenates and detergent extracts prepared fromXenopuscentral nervous system was evaluated in 50 mMTris‐citrate buffer, pH 7.0. In membrane fragment preparations, [3H]kainate bound with aKDof 54.4 nMto a large number of sites (Bmax= 27.8 pmol/mg of protein). Up to 80% of the total number of membrane‐bound binding sites were solubilised using the nonionic detergentn‐octyl‐β‐d‐glucopyranoside. Values for theKDof [3H]kainate for solubilised binding sites were 46.0 nMand 53.6 nMderived from equilibrium and kinetic binding experiments, respectively. Competitive binding studies revealed that a variety of ligands had similarKivalues in both membranes and solubilised extracts, with domoate and kainate being the most potent inhibitors of [3H]kainate binding. The dissociation rate of [3H]kainate from solubilised binding sites was 0.022 min–1. The binding component migrated in sucrose density gradients in a single 8.6S peak. These results demonstrate that the kainate receptor inXenopuscentral nervous system, although similar to the [3H]kainate binding site from goldfish brain, differs in a number of important respects. In particular, the slower dissociation rate and higher affinity of [3H]kainate suggest thatXenopusprovides the most convenient model system yet investigated for biochemical analysis of kai

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10894.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:The effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the intracellular accumulation of inositol phosphates and on cytosolic free Ca2+concentrations were studied in rat PC12 pheochromocytoma cells. Both NGF and EGF potentiate in these cells the increase in the accumulation of inositol phosphates that is elicited by bradykinin and carbachol. A corresponding potentiation was also found for the agonist‐induced increase of cytosolic Ca2+concentrations. The effect of NGF, but not that of EGF, is abolished when the cells are preincubated with 5′‐deoxy‐5′‐methylthioadenosine, an inhibitor ofS‐adenosylhomocysteine hydrolase. These results suggest that an increased response to hormones, which act via phosphoinositide‐derived second messengers, may be important in the mechanism of action

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10895.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Treatment of neonatal, but not adult, rats with glucocorticoids decreases the rise in pineal serotoninN‐acetyltransferase activity upon stimulation with β agonists. Pineals in organ culture and exposed to steroids also show a dose‐dependent decrement in response to β agonists which increases with steroid exposure time. Pineals from neonatal and adult animals are equally sensitive. The effects of steroids on pineals in organ culture appear to be reversible, and the order of potency of different steroids differs from that observed when steroids are administered in vivo. Both in vitro and in vivo steroids appear to act at a site after cyclic AMP generation. Hydroxyindole‐O‐methyltransferase activity in the adult pineal does not appear affected by steroid

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10896.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Incubation of synaptosomes from rat brain withdl‐2‐amino‐5‐phosphonovalerate (APV) stimulated an increased release of dopamine, and this effect was strictly dependent on the extrasynaptosomal calcium level. APV increased biosynthesis of dopamine from tyrosine by 30%, whereas monoamine oxidase activity was inhibited by 30%. When synaptosomes were incubated with radioactive dopamine, APV caused a large decrease in incorporation of label into 3,4‐dihydroxyphenylacetic acid but greatly increased incorporation into norepinephrine and itsN‐methyl derivatives. Quantification of dopamine and its metabolites in synaptosomes, using electrochemical detection, indicated that the presence of APV resulted in changes in the absolute levels of the aforementioned dopamine metabolites similar to the changes in radiolabel incorporation. Omission of Ca2+from the extrasynaptosomal medium greatly diminished the APV‐induced changes in catecholamine metabolism. The metabolic changes appear to largely result from an increased intrasynaptosomal Ca2+level due to the APV‐induced increase in calcium permeability of the

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10897.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Two dynorphin‐degrading cysteine proteases, I and II, were extracted with Triton X‐100 from neuroblastoma cell membrane, isolated from accompanying dynorphin‐degrading trypsin‐like enzyme by affinity chromatography on columns of soybean trypsin inhibitor‐immobilized Sepharose andp‐mercuribenzoate–Sepharose, and separated by ion‐exchange chromatography on diethylaminoethyl (DEAE)‐cellulose and TSK gel DEAE‐5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes were inhibited byp‐chloromercuribenzoate,N‐ethylmaleimide, and high‐molecular‐weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E‐64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1–17) at the Arg6‐Arg7bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1–17) at the Lys11‐Leu12bond and the Leu12‐Lys13bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg‐Arg doublet, when susceptibilities of various peptides containing paired basic residues w

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10898.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Previous reports from several laboratories have demonstrated the presence of many lipid‐metabolizing enzymes in myelin, including all the enzymes needed to convert diacylglycerol to phosphatidylcholine and phosphatidylethanolamine. Axonal transport studies had suggested the presence of additional enzymes which incorporate acyl chains into specific phospholipids of myelin. We report here evidence for one such group of enzymes, the acyl‐CoA:lysophospholipid acyltransferases. At the same time, activity of acyl‐CoA:sn‐glycerol‐3‐phosphate acyltransferase was negligible in myelin. Oleoyl‐CoA and arachidonoyl‐CoA were both active substrates for transfer of acyl chains to lysophosphatidylcholine and lysophosphatidylinositol. Activity in myelin varied from 7 to 19% of microsomal activity, values well above the likely level of microsomal contamination as judged by microsomal markers. Additional evidence for a myelin locus came from assays at sequential stages of purification and from mixing experiments. Arachidonoyl‐CoA was somewhat more reactive than oleoyl‐CoA toward lysophosphatidylcholine; the myelinKmfor these two CoA derivatives was 98 μMand 6.6 μM, respectively. Activity with lysophosphatidylinositol as substrate was approximately 40% of that with lysophosphatidylcholine in myelin, whereas activities with lysophosphatidylethanolamine and lysophosphatidylserine w

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10899.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY