摘要:

Abstract:Antibodies were prepared to mammalian CNS neurofilament proteins (NFPs) and the antibody specificities were compared using a sensitive immunoblotting method. This procedure was used to detect and characterize cross‐reactive proteins and their degradation products in neurofilament preparations. NFPs were prepared by axon flotation. Rabbits were immunized with 200,000, 140,000 and 70,000 NFPs (200K, 140K and 70K) that had been electrophoretically purified by polyacrylamide gel electrophoresis (PAGE). By immunohistofluorescence it was shown that all antisera stained similar filamentous structures in rat cerebellar neurons. By use of a horseradish peroxidase‐conjugated indirect antibody procedure, however, differences were detected in the cross‐reactivities of the antisera to rat NFPs, separated by PAGE and electrophoretically transferred to nitrocellulose membranes. Each antiserum exhibited strong binding to the homologous NFP and thus, was suitable for the detection of cross‐reactive polypeptides and proteolytic degradation products derived exclusively from the individual NFPs. Anti‐200K, anti‐140K, or anti‐70K was applied to overloaded two‐dimensional nitrocellulose blots of NFPs prepared by axon flotation. Each of the three sera detected a group of unique nonoverlapping polypeptides, some of which were identified as NFP degradation products. A different group of polypeptides was cross‐reactive with antiserum to purified glial fibrillary acidic protein. The immunostaining of polypeptides on nitrocellulose was far more sensitive for detecting NFP degradation products than was staining polyacrylamide gels with Coomassie blue. Tilers for the antisera were two to three orders of magnitude higher with the immunoblotting procedure than with immunohistologic methods. The sensitivity and the specificity of the described methods suggest their usefulness for examining proteolytic cleavage products of NFPs under a vari

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb11283.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Arachidonic acid, a major polyunsaturated fatty acid of membrane phospholipids in the CNS, reduced the high‐affinity uptake of glutamate and γ‐aminobutyric acid (GABA) in both rat brain cortical slices and synaptosomes. α‐Aminoisobutyric acid uptake was not affected. Intrasynaptosomal sodium was increased concomitant with decreased (Na++ K+)‐ATPase activity in synaptosomal membranes. The reduction of GABA uptake in synaptosomes could be partially reversed by α‐tocopherol. The inhibition of membrane‐bound (Na++K+)‐ATPase by arachidonic acid was not due to a simple detergent‐like action on membranes, since sodium dodecyl sulfate stimulated the sodium pump activity in synaptosomes. These data indicate that arachidonic acid selectively modifies membrane stability and integrity associated with reductions of GABA and glutamate uptake and of (Na++ K

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb11284.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Rat brain capillaries exhibit concentrative uptake ofl‐proline. The uptake is mediated by two saturable systems, one with aKmof 0.11 mMand another with aKmof 5.9 mM.Entry also occurred by diffusion, especially at high substrate concentrations. The saturable high‐affinity system is sodium‐dependent, with aKmfor sodium of 36 mM.Proline uptake is not inhibited by lysine, but is inhibited by phenylalanine, glycine and leucine. α‐Methylaminoisobutyric acid (MeAIB), a model for sodium‐requiring transport systems, is a competitive inhibitor of the low‐Kmsystem. b‐2‐Aminobicyclo‐[2, 2, 1]‐heptane‐2‐carboxylic acid (BCH), a model for nonsodium‐dependent transport, however, als

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb11285.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Incubation of rat brain synaptic membranes under phosphorylating conditions (i.e., in the presence of Mg2+, ATP and cyclic AMP) leads to a loss in muscarinic acetylcholine receptors, detectable as specific binding of the muscarinic antagonist L‐[3H]quinuclidinyl benzilate. A role for protein phosphorylation in this receptor loss is indicated by the finding that 5′‐adenylyl imidodiphosphate, a nonhydrolysable analogue of ATP, does not support receptor loss. Furthermore, receptor loss is inhibited by adenosine and 2‐deoxyadenosine, both of which inhibit protein kinase activity. The loss of muscarinic receptors is calmodulin dependent and it has been demonstrated here that this requirement is probably at the level of calmodulin‐dependent phosphorylation. An investigation of the effects of phosphorylation on the binding of the agonist carbachol to synaptic membranes from the cortex and cerebellum demonstrated that phosphorylation altered the relative proportions of the super‐high‐, high‐ and low‐affinity binding sites. The results were consistent with an apparent conversion of high‐ into super‐high‐affinity sites. In the presence of 5′‐guanylyl imidodiphosphate, agonist binding demonstrated the properties expected of a population of largely low‐affinity sites. This conversion of super‐high‐ and high‐affinity sites into low‐affinity sites by 5′‐guanylyl imidodiphosphate w

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb11286.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Tryptophan uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on the Na+gradient [Na+] outside>[Na+] inside and is maximal when both Na+and Cl−are present. The uptake represents transport into an os‐motically active space and not a binding artifact, as indicated by the effect of increasing the medium osmo‐larity. The uptake of tryptophan is stimulated by a membrane potential (interior negative) as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m‐chlorophenylhydrazone and anions with different permeabilities. Kinetic data show that tryptophan is accumulated by two systems with different affinities. Ouabain, an inhibitor of Na+, K+‐activated ATPase, does not affect tryptophan transport. The uptake of tryptophan is inhibited by high concentrations of phenylalanine, tyrosine, leucine and 3, 4‐dihydroxyph

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb11287.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:We adapted a method, originally described by Israel et al. (1976) for the preparation of cholinergic nerve endings fromTorpedo, to deal with a larger quantity of electric tissue. We followed the distribution of acetylcholine (ACh), ATP, acetylcholine receptor (AChR), choline acetyltransferase (ChAT), ouabainresistant and ‐sensitive ATPase, lactate dehydrogenase (LDH) and acetylcholinesterase (AChE) and obtained a nerve ending fraction, without detectable contamination by postsynaptic components. This preparation consisted of closed structures of 1–5 μm diameter, containing synaptic vesicles. It had the capacity to synthetize and release ACh. This preparation is therefore quite suitable for biochemical analysis of presynaptic elements. We particularly investigated its content of AChE: it consists exclusively of the 6S dimeric, hydrophobic form of the enzyme. This enzyme is enriched in the nerve ending preparation, by a factor higher than that obtained for ChAT. The yields obtained for the two enzymes suggest that the hydrophobic 6S AChE form may be mostly presynaptic inTorpedoelectric organs. We characterized this form as a membrane‐bound, externally active enzyme in the nerve ending preparation. It may thus participate in the hydrolysis of extracellularly liberated AChE and its abundance suggests that presynaptic AChE could play an essential role in cholinergic transmission inTorpedoelectric organs and perhaps also in other cholinergic sy

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb11288.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:The effect of a stressful manipulation on the metabolism of γ‐aminobutyric acid (GABA) in the rat brain was studied. Application of an immobilized stress to animals induced a significant increase in the striatal and hypothalamic GABA contents without affecting those in other central structures examined. It was also found that the increase in striatal GABA level preceded that in the hypothalamus. This increase in steady‐state levels of GABA in the striatum and hypothalamus disappeared at 12 h after the termination of the application of stress for 3 h, which exhibited a maximal stimulatory action on the GABA contents in both central areas. The activity ofl‐glutamic acid decarboxylase was found to be significantly elevated in the striatum and hypothalamus following the stress application with a concomitant decrease in the content of L‐glutamic acid, which is converted to GABA by the catalytic action of the latter enzyme. Thein vivoturnover of GABA in the brain was estimated by taking advantages of the postmortem accumulation of GABA following decapitation and of the selective inhibitory action of a low dose of aminooxyacetic acid on the GABA degrading systems, respectively. Analysis using these two different methods revealed that the cerebral turnover of GABAin vivowas not significantly altered under stressful situations despite of the increase in its steady‐state level. These results suggest that central GABA system may respond to the input of painful stimuli resulting from the application of a severe physical and psychological stressor, in addition to the well‐known functional alterations in catecholamine neurons. The functional significance of these alterations in the central GABA neurons is al

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb11289.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Stores of methionine‐enkephalin were labelled on theN‐terminal by incubation of whole brain slices with [3H]tyrosine (10 °Ci/ml). The3H radioactivity corresponding to the position of authentic Met‐enkephalin after extraction on Amberlite XAD2and separation by thin‐layer chromatography was taken as an index of synthesis. Maximal incorporation of the labelled tyrosine into Met‐enkephalin was attained after 4 h of incubation at 37°C and was inhibited in the presence of 10 μMcycloheximide. Isolated nerve terminals failed to incorporate any [3H]tyrosine. The labelled compound had opiatelike activity and consisted of the same five amino acids as an authentic standard. Incubations with leucine aminopeptidase indicated that the labelled tyrosine was on theN‐terminus and removal of this tyrosine resulted in loss of opiate‐like activity. The incorporation of [14C]glycine, selected as an alternative precursor, was consistent withde novosynthesis and notN‐terminal exchange. A radioimmunoassay was also used to quantify the amount of labelled Met‐enkephalin. KCl (50 mM) elicited a Ca2+‐dependent release of the synthesised [3H]Met‐enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met‐enkephalin radioimmunoactivity paralleled that of [3H]met‐enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release and its effect was pa

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb11290.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:Glucocorticoids, cholera toxin and high plating density all increase the activity of tyrosine 3‐monooxygenase (TH) in cultured PC12 pheochromocytoma cells. Glucocorticoids increase enzyme activity in cells treated with cholera toxin and in cells grown at high plating density. Glucocorticoids also increase the content of stored catecholamines in the cells. In cells cultured under routine conditions, glucocorticoids primarily increase the stores of dopamine. The addition of ascorbate to the culture medium increases the storage of norepinephrine in both steroid‐treated and untreated cells. Incubation of the cells in media containing 56 nMK+causes the release of the same percentage of stored dopamine from steroid‐treated as from untreated cells. Steroid‐treated cells contain more dopamine than do untreated cells and therefore, in response to high K+, the steroid‐treated cells secrete more dopamine than do untreated cells. We conclude that the activity of tyrosine 3‐monooxygenase in PC12 cells can be regulated by several distinct mechanisms; that glucocorticoids cause a coordinate increase in TH activity and in catecholamine storage; that steroids increase the storage of catecholamines in a releasable pool; and that the steroid‐induced increase in catecholamine storage may result in increased secretion of catecholamines from steroid

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb11291.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

Abstract:A biochemical method is described for the simultaneous quantitative estimation of unidirectional blood‐brain amino acid influx and protein biosynthesis in individual structures of the rat brain. The method involved a double labeling experiment started by the administration of [14C]carboxyl‐labeled amino acids and terminated 2 min after infusion of3H‐labeled amino acids, each at tracer quantities, the total labeling period being 45 min. Specific radioactivities of14C‐ or3H‐labeled phenyl‐alanine, tyrosine, leucine, isoleucine and valine were determined in plasma and in small brain tissue samples for free amino acids, aminoacyl‐tRNAs and proteins. Amino acids were converted to their corresponding 5‐dimethylamino‐naphthalenesulfonyl (Dns, dansyl) derivatives and separated on HPLC C18reversed‐phase columns isocratically according to a newly developed optimizing procedure. The order of influx values between the neutral amino acids in relation to each other was Leu>Tyr>Ile>Phe>Val in every structure examined. Although aminoacylation of tRNAs was found to proceed to a comparable degree for neutral amino acids in all regions investigated, the specific radioactivity of amino acids attached to tRNAs differed substantially from that in the free amino acid pool, especially for leucine and valine. The results indicate the necessity of aminoacyl‐tRNA determinations for tracer incorporation studies in protein synthesis analysis. Relative protein synthesis rates in the halothane‐anesthetized rat were determined to be 30 and 67–91 pmol total amino acid incorporation/min/mg tissue for white and

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1983.tb11292.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY