摘要:

Abstract:The absolute amounts of precursor to ribosomal RNA (pre‐rRNA) and ribosomal RNA (rRNA) in isolated rat brain neuronal and oligodendroglial nuclei were determined. The amount of the major pre‐rRNA and rRNA species in neuronal nuclei was about twofold higher than in oligodendroglial nuclei. The relative rate of pre‐rRNA synthesis in vivo was 2.3‐ to 2.7‐fold higher in neuronal as compared with oligodendroglial nuclei. This corresponds to a 2.7‐fold higher activity of the “template‐bound” RNA polymerase I in isolated neuronal nuclei, whereas the activity of the “free” enzyme in both neuronal and glial nuclei was almost identical. The higher transcription rates of rRNA genes correlated with the markedly more prominent fibrillar component in neuronal nucleoli. The turnover times of the major pre‐rRNA and rRNA species in neuronal and oligodendroglial nuclei were similar, except for 45S pre‐rRNA, which turned over at an 1.5‐fold slower rate in neuronal nuclei. The relative rates of processing of pre‐rRNA and of nucleocytoplasmic transport of rRNA in neuronal cells were 2.7‐fold higher than in oligodendroglial cells and corresponded to the differences in rRNA gene transcription rates. The established ribosome formation features correlated with an abundant (neurons) or exceedingly scarce (oligodendrocytes) nucleolar granular component. The turnover rate of cytoplasmic ribosomes in rat brain neurons was twofold slower than in oligodendrocytes, largely because of the about fivefold higher amount of ribosomes in the cytoplasm of neurons. We conclude that ribosome formation and turnover in neuronal and oligodendroglial cells are adapted to the protein synthetic levels in

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1985.tb10521.x

出版商:Blackwell Publishing Ltd    

年代:1985

数据来源: WILEY

摘要:

Abstract:Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome‐enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+‐dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40–80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release excitatory amino acids stimulated by high K+concentrations. Kainic acid at 1 mMshowed a completely opposite effect on the release of exogenously accumulated D‐[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+‐stimulated release. Kainic acid at 0.1 mMhad no clear‐cut effect, whereas at 0.01 mMit caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier‐mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high‐affinity uptake of L‐glutamate and D‐aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kaini

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1985.tb10522.x

出版商:Blackwell Publishing Ltd    

年代:1985

数据来源: WILEY

摘要:

Abstract:Rat brain striatum slices were incubated with [3H]choline, perfused with a physiological buffer, and stimulated by perfusion with a K+‐enriched buffer for 2 min. The tritium overflow evoked by K+was decreased by 5‐hydroxytrytamine (serotonin, 5‐HT) (maximal inhibition 10–6M). This effect of 5‐HT was mimicked by several agonists (5‐methoxytryptamine,N,N‐dimethyltryptamine, bufotenin) and blocked by serotonergic antagonists (methiothepin, methysergide, cinanserin) but not by haloperidol; methiothepin and methysergide alone slightly increased the K+‐evoked overflow of tritium (3H). Inhibition of the tritium release by 5‐HT was not suppressed in the presence of tetrodotoxin (TTX) (10–6M) These results suggest that 5‐HT tonically inhibits acetylcholine (ACh) release from striatal cholinergic neurons by acting on a presynaptic receptor localized on

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1985.tb10523.x

出版商:Blackwell Publishing Ltd    

年代:1985

数据来源: WILEY

摘要:

Abstract:The influence of varying doses of streptozotocin and preventive insulin treatment on phospholipid metabolism in sciatic nerve in vitro from diabetic rats was studied. Animals were given 30, 45, and 60 mg/kg injections of streptozotocin and 10 weeks later nerves were removed and incubated in the presence of [32P]orthophosphate. The quantity of isotope incorporated into phosphatidylinositol‐4,5‐bisphosphate (PIP2) was progressively greater with increasing drug dosage, whereas uptake of label into other phospholipids was unchanged. Rats were made diabetic and within 72 h were implanted with long‐acting, insulin‐containing osmotic minipumps and the incorporation of [32P]orthophosphate into phospholipids of intact and epineurium‐free nerves was examined 8 weeks later. For whole nerve, increased labeling in nerves from diabetic animals occurred only in PIP2and phosphatidylinositol‐4‐phosphate (PIP) and was completely prevented by insulin treatment. Isotope incorporation into polyphosphoinositides was also markedly elevated (100%) in desheathed diabetic nerves, but not in nerves from insulin‐treated animals. Other phospholipids in epineurium‐free nerves displayed some rise in isotope uptake, but the increases were not prevented by insulin treatment and appeared unrelated to hyperglycemia. Morphological examination of nerves extended previous findings that prolonged insulin treatment produces axonal degeneration. These observations indicate that abnormal nerve polyphosphoinositide metabolism is at least in part a consequence of hyperglycemia. The metabolic alterations may be intimately involved in reduced nerve conduction velocity, which is characteristic of di

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1985.tb10524.x

出版商:Blackwell Publishing Ltd    

年代:1985

数据来源: WILEY

摘要:

Abstract:In spite of biochemical and autoradiographic evidence for glucocorticoid binding sites in the spinal cord (SC), events occurring after the preliminary step of hormone binding were not studied. In this investigation, we have examined the transformation (activation) of the cytosolic receptor coupled to [3H]dexamethasone (DEX) and the in vivo interaction of adrenal hormone [corticosterone (CORT)]with purified nuclei from the SC, in addition to the CORT content of the SC before and after stress. Binding of [3H]DEX in the SC was 40% lower than in the hippocampus (HC), although theKDvalues were comparable. Transformation of [3H]DEX‐receptor complexes in the cytosol was demonstrated by diethylaminoethane‐cellulose chromatography, by DNA‐cellulose binding, and by a combined minicolumn procedure including hydroxyapatite in addition to the last two techniques for separation of transformed, nontransformed, and meroreceptor complexes. In all these situations, SC glucocorticoid binding sites behaved similarly to those in the HC. Nuclear uptake of a tracer dose of [3H]CORT was much lower in the SC than in the HC; nuclear retention of CORT was more easily detected by radioimmunoassay after injection of 1 mg of CORT into adrenalectomized rats. Substantial amounts of CORT, which increased in level after stress, were measured in five regions in the SC, with higher concentrations in the cervical regions. These studies suggest that although SC and HC receptors show similar properties in vitro, differences emerged at the level of nuclear uptake in vivo, in that glucocorticoid action in the SC was similar to that in the optic nerve, where receptors seem to be localized mostly in glial

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1985.tb10525.x

出版商:Blackwell Publishing Ltd    

年代:1985

数据来源: WILEY

摘要:

Abstract:Leptinotoxin‐h (LPTx), a neurotoxin (otherwise designated β‐leptinotarsin‐h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle,Leptinotarsa haldemani, by a simplification of the procedure originally developed by Crosland et al. [Biochemistry23, 734–741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca2+‐containing Ringer medium, at concentrations in the 10−11‐10−10Mrange, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage‐dependent Na+and Ca2+channels (tetrodotoxin or verapamil); (b) large45Ca influx: and (c) increased free cytosolic Ca2+concentration. These latter two effects were unaffected by verapamil. In Ca2+‐free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca2+is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono‐ and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca2+channel as the mechanism of action of LPTx. The effects of the toxin resemble those of α‐latrotoxin (α‐LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx do

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1985.tb10526.x

出版商:Blackwell Publishing Ltd    

年代:1985

数据来源: WILEY

摘要:

Abstract:Guinea pig brain cortex synaptosomes and neurosecretory PC12 cells were loaded with [3H]3,4‐di‐hydroxyphenylethylamine ([3H]DA, [3H]dopamine) and then exposed to leptinotoxin‐h (LPTx) (purified and partially purified preparations, obtained from the hemo‐lymph ofLeptinotarsa haldemani). In a Ca2+‐containing Ringer medium the toxin induced prompt and massive release of the neurotransmitter. Half‐maximal effects were obtained at concentrations estimated of approximately 3 × 10−11Mfor synaptosomes, and 1.5 × 10−10Mfor PC12 cells. Release responses in the two experimental systems investigated were dependent to different extents on the Ca2+concentration in the medium. In synaptosomes clear, although slow, release of [3H]DA was elicited by the toxin even in Ca2+‐free, EGTA‐containing medium, provided that high (in the 10−10Mrange) concentrations were used; near‐maximal responses were observed at 10−5MCa2+. In contrast, the toxin‐induced release from PC12 cells was appreciable only at 3 × 10−5MCa2+, and was maximal at 2 × 10−4Mand above. In both synaptosomes and PC12 cells Sr2+and Ba2+could substitute for Ca2+; Co2+was inhibitory, whereas Mn2+failed to modify the release induced by the toxin in Ca2+‐containing medium. Organic blockers of the voltage‐dependent Ca2+channel (verapamil and nitrendipine) and calmodulin blocking drugs (trifluoperazine and calmidazolium) failed to inhibit the toxin‐induced release of [3H]DA. LPTx induced profound morphological effects. Synaptosomes treated in the Ca2+‐containing medium exhibited fusion of synaptic vesicles, formation of numerous infoldings and large cisternae, and alterations of mitochondria. In the Ca2+‐free medium the effects were similar, except that their appearance was delayed, and mitochondria were well preserved. Swelling was observed in PC12 cells, accompanied by enlargement of the Golgi area, accumulation of multivesicular bodies, mitochondrial alterations, and decreased number of secretion granules (Ca2+‐containing medium). Morphometric analyses revealed a good correlation between the decrease of both synaptic vesicles (synaptosomes) and neurosecretory granules (PC12 cells), and the release of [3H]DA measured biochemically. This is a good indication that the release effect of the toxin is due to stimulation of exocytosis. Taken as a whole, these results confirm the similarity of the effects of LPTx with α‐latrotoxin of the black widow spider venom, mentioned in the companion article. However, differences in effect and target specificity suggest that the two toxins are specific to separate binding sites. Because of its profound and exclusively presynaptic effects LPTx promises to be an interesting tool for the study of the

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1985.tb10527.x

出版商:Blackwell Publishing Ltd    

年代:1985

数据来源: WILEY

摘要:

Abstract:A neuroblastoma × Chinese hamster embryonic brain explant hybrid cell line (NCB‐20) expressed 5‐hydroxytryptamine (5‐HT1) receptors, linked to adenylate cyclase, which closely resembled 5‐HT1receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5‐HT was only 150 nMcompared to 5 nMin membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB‐20 cells produced by 5‐HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1(PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5‐HT. The elevation of cyclic AMP levels by either 5‐HT or PGE1was reversed by [D‐Ala2,D‐Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4‐dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5‐HT or PGE1alone. When the DADLE exposure time was increased to 48 h, 5‐HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5‐HT resulted from an increase in receptor affinity for 5‐HT (from aKDof 150 ± 20 nMto one of 20 ± 7 nM), with a reduction in the number of apparent binding sites. The 5‐HT supersensitivity observed in NCB‐20 cells may be a good model for neurotransmitter interactions that produce desensitization or fac

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1985.tb10528.x

出版商:Blackwell Publishing Ltd    

年代:1985

数据来源: WILEY

摘要:

Abstract:Cultured NCB‐20 hybrid cells express adenylate cyclase‐coupled receptors for 5‐hydroxytryptamine (5‐HT) that correspond biochemically and pharmacologically to 5‐HT1receptors in rodent brain membrane preparations, apart from a much‐reduced affinity for 5‐HT (160 nMcompared to<5 nMin brain). Since NCB‐20 cells also differ from rodent brain both qualitatively and quantitatively in their ganglioside composition, the effects of exogenously added gangliosides on the affinity of the 5‐HT1receptor for 5‐HT were tested. Both GM1ganglioside (the cholera toxin receptor) and tetrasialoganglioside GQ1bproduced a 10‐fold increase in receptor affinity for [3H]5‐HT, measured by binding studies. All gangliosides, at submicromolar concentrations, resulted in significantly reduced EC50values for 5‐HT‐mediated elevation of intracellular cyclic AMP levels. GQ1bhad the capacity to most dramatically enhance the potency of 5‐HT in mediating increases in cyclic AMP levels. Gangliosides had no effect on the potency of DADLE or 3,4‐dihydroxyphenylethylamine (dopamine)‐mediated depression of cyclic AMP levels, suggesting some specificity for 5‐HT. Our data are interpreted as implying a specific role for polysialogangliosides in modulating the affinity of the 5‐HT1receptor and the coupling of the 5‐HT1receptor‐guanine nucleotide

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1985.tb10529.x

出版商:Blackwell Publishing Ltd    

年代:1985

数据来源: WILEY

摘要:

Abstract:Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine‐bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5‐4864, Ro5‐3453, Ro11–6896, and Ro5–3438 (theKDvalues are in the 10−9Mrange). However, these antibodies have low affinities for the benzodiazepine receptor inverse agonists (β‐carbolines) and antagonists (Ro15–17

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1985.tb10530.x

出版商:Blackwell Publishing Ltd    

年代:1985

数据来源: WILEY