摘要:

Abstract:The regulation of μ‐(MOR) and δ‐opioid receptor (DOR) after chronic cocaine administration has been studied. Male Sprague‐Dawley rats were treated for 3 days with saline and cocaine (50 mg/kg/day) delivered by osmotic minipump. Expression of MOR and DOR mRNA in olfactory bulb, nucleus accumbens, and caudate‐putamen (caudal and rostral parts) was estimated using quantitative competitive PCR assays after reverse transcription. No changes in the levels of mRNA for DOR were detected after exposure to cocaine in the brain regions examined. A significant increase in the level of MOR mRNA was detected in nucleus accumbens after 3 days of cocaine treatment. In caudate‐putamen and olfactory bulb, no change in MOR mRNA was observed after cocaine administration. Both SCH 23390 and eticlopride, selective antagonists of D1‐ and D2‐dopamine receptors, respectively, blocked this cocaine‐induced up‐regulation of MOR mRNA in nucleus accumbens. We suggest that endogenous opioid systems in nucleus accumbens, the brain region specifically associated with the reinforcing properties of addictive drugs, are regulated by dopaminergic mechanisms and influenced b

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66020443.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The posttranscriptional regulation of glucose transporter GLUT1 gene expression may be mediated by specific interactions of cytosolic proteins and regulatorycis‐elements within the untranslated regions (UTRs) of the GLUT1 mRNA. These putativecis/transinteractions were examined in the present studies with RNase T1 protection assays using32P‐labeled GLUT1 3′‐UTR prepared from transcription plasmids and cytosolic proteins from C6 rat glioma cells. RNase T1 mapping studies localized acis‐element to nucleotides 2,170–2,207 on the bovine GLUT1 mRNA 3′‐UTR. Ultraviolet cross‐linking of RNA/protein complexes identified two complexes having molecular masses of 88 and 44 kDa. Competition studies with synthetic RNA and oligodeoxynucleotides showed the 88‐kDa complex reacted with nucleotides 2,180–2,197 and that the 44‐kDa complex reacted with sequences within nucleotides 1,717–2,132 of the bovine GLUT1 mRNA. The GLUT1 3′‐UTR between nucleotides 2,100 and 2,300 was generated by polymerase chain reaction and subcloned at a uniquePf/MI site within the 3′‐UTR of a luciferase gene within the mammalian expression vector pGL2. Transfection of C6 rat glioma cells with the luciferase expression vector containing this portion of the GLUT1 3′‐UTR resulted in a sixfold increase in luciferase gene expression in C6 cells. The identification of thesecis/transmechanisms provides support for the hypothesis that the posttranscriptional regulation of GLUT1 gene expression may be mediated by the interaction of specific cytosoli

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66020449.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Astrocytes have been shown to express endothelin (ET) receptors functionally coupled, via different heterotrimeric G proteins, to several intracellular pathways. To assess the relative contribution of each subtype in the astrocytic responses to ET‐1, effects of BQ123, an antagonist selective for the ET receptor subtype A (ETA‐R), and IRL1620, an agonist selective for the ET receptor subtype B (ETB‐R), were investigated in primary cultures of rat astrocytes. Binding experiments indicated that the ETB‐R is the predominant subtype in these cells. Inhibition of forskolin‐stimulated cyclic AMP production was observed under ETB‐R stimulation.Bordetella pertussistoxin (PTX) pretreatment completely abolished this effect, indicating that this pathway is coupled to the ETB‐R via Giprotein. Increases of tyrosine phosphorylation of cellular proteins, stimulation of mitogen‐activated protein kinase (MAPK), and DNA synthesis were also found to be mediated by the ETB‐R, but through PTX‐insensitive G protein. IRL1620‐induced MAPK activation involved the adapter proteins Shc and Grb2 and the serine/threonine kinase Raf‐1. This study reveals that the various effects of ET‐1 in astrocytes are mediated by the ETB‐R, which couples to multiple signaling pathways

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66020459.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Rat C6 glioma synthesizes a low basal level of interleukin‐6 (IL‐6). Stimulation with 10 µg/ml of lipopolysaccharide (LPS) and induction of differentiation with 1 mM N6,O2′‐dibutyryl cyclic AMP (dbcAMP) for 48 h increased the secreted activity to 400 and 800 U/ml, respectively. An LPS stimulation of dbcAMP‐differentiated cells strongly enhanced the secreted activity. Depending on the dbcAMP concentration, the cell number, and the stimulation time, the secreted IL‐6 level increased up to 120,000 U/ml. After 48 h of costimulation with 10 µg/ml of LPS and 1 mMdbcAMP, northern blotting and immunoassay demonstrated an eightfold increase in IL‐6 mRNA concentration and IL‐6 immunoreactivity, whereas titration of the biological activity indicated a 100‐fold increase in the secreted IL‐6 activity. The enhanced secretion of IL‐6 is correlated with the induction of differentiation. Chromatography on heparin‐Sepharose and on DEAE‐5PW separated the secreted activity into several fractions, indicating that they differ in heparin affinity and charge either by posttranslational modifications or by binding to a carrier protein. Each of the partially purified IL‐6‐like activities could be neutralized by an anti‐murine IL‐6 antibody. Our observations demonstrate that in vivo inflammatory signals can trigger astrocytes and their precursors to secrete substantially different levels of immunoregulatory cytokines depending

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66020466.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Impaired energy metabolism may contribute to the pathogenesis of late‐onset neurodegenerative disorders such as Alzheimer's disease by increasing neuronal vulnerability to excitotoxic damage through the NMDA receptor. The effects of metabolic impairment on the striatum have been extensively examined, but relatively little is known regarding the vulnerability of the hippocampus. To examine the effect of metabolic impairment on the hippocampal formation, malonate (0.25–2.5 µmol), a reversible inhibitor of succinate dehydrogenase, was administered by stereotaxic injection into the hippocampus of male Sprague‐Dawley rats. Neuronal loss was assessed by Nissl stain, and immunocytochemistry was used to examine cytoskeletal disruption. Malonate produced a dose‐dependent lesion in which CA1 pyramidal neurons were most vulnerable, followed by CA3 and dentate gyrus. Cytoskeletal alterations included the loss of microtubule‐associated protein 2 (MAP2) and dendritic MAP1B immunoreactivity, whereas axonal MAP1B and τ proteins were relatively spared. Spatially and temporally correlated with the loss of MAP2 was an increase in the immunoreactivity of calpain‐cleaved spectrin. A similar pattern of neuronal damage and cytoskeletal disruption was produced by intrahippocampal injection of quinolinate (0.1 µmol), an NMDA agonist. Although these results are consistent with the hypothesis that metabolic impairment results in excitotoxic death, MK‐801 (dizocilpine maleate), a noncompetitive NMDA receptor antagonist, did not attenuate the lesions produced by malonate but was effective against quinolinate. The results suggest that NMDA receptor activation is not required for malonate‐induced damage in the hip

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66020474.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Somatostatin (SS) is a neuropeptide that is distributed in various regions of the CNS, where it may act as a neurotransmitter or neuromodulator. SS produces multiple effects in the CNS through interactions with membrane receptors. In particular, SS inhibits various secretory responses in different cell types. In the present study, we have investigated the effects of exogenous application of SS on the intracellular free Ca2+concentration ([Ca2+]i) in PC12 cells, a rat pheochromocytoma cell line. SS did reduce the magnitude of the secondary, maintained Ca2+influx brought about by K+depolarization. Similar effects were obtained with the application of SS analogues, such asd‐Trp8‐SS,d‐Trp8‐d‐Cys14‐SS, CGP‐23996, and SMS‐201995. In addition, treatment with cyclo‐SS, a SS antagonist, did not alter [Ca2+]i. Experiments with selective blockers of different voltage‐dependent Ca2+channels, such as methoxyverapamil (D600) and Ω‐conotoxin GVIA, demonstrated that the effects of SS on [Ca2+]iwere mediated by voltage‐dependent Ca2+channels of the L type. Control experiments with a membrane potential indicator, i.e., the fluorescent dye bisoxonol, excluded that SS influenced the level of the membrane potential. SS effects on PC12 cells suggest the possibility that this neuropeptide plays a role in the modulation of cell functional activit

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66020485.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:To investigate the route of axonal Ca2+entry during anoxia, electron probe x‐ray microanalysis was used to measure elemental composition of anoxic tibial nerve myelinated axons after in vitro experimental procedures that modify transaxolemmal Na+and Ca2+movements. Perfusion of nerve segments with zero‐Na+/Li+‐substituted medium and Na+channel blockade by tetrodotoxin (1 µM) prevented anoxia‐induced increases in Na and Ca concentrations of axoplasm and mitochondria. Incubation with a zero‐Ca2+/EGTA perfusate impeded axonal and mitochondrial Ca accumulation during anoxia but did not affect characteristic Na and K responses. Inhibition of Na+‐Ca2+exchange with bepridil (50 µM) reduced significantly the Ca content of anoxic axons although mitochondrial Ca remained at anoxic levels. Nifedipine (10 µM), an L‐type Ca2+channel blocker, did not alter anoxia‐induced changes in axonal Na, Ca, and K. Exposure of normoxic control nerves to tetrodotoxin, bepridil, or nifedipine did not affect axonal elemental composition, whereas both zero‐Ca2+and zero‐Na+solutions altered normal elemental content characteristically and significantly. The findings of this study suggest that during anoxia, Na+enters axons via voltage‐gated Na+channels and that subsequent increases in axoplasmic Na+are coupled functionally to extraaxonal Ca2+import. Intracellular Na+‐dependent, extraaxonal Ca2+entry is consistent with reverse operation of the axolemmal Na+‐Ca2+exchanger, and we suggest that this mode of Ca2+influx plays a general role in

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66020493.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Incubation withl‐DOPA induced a rise in GSH level in cultures of fetal rat mesencephalon, mouse neuroblastoma (Neuro‐2A), human neuroblastoma (SK‐N‐MC), pig kidney epithelial cells (LLC‐PK1), and glia from newborn rat brain, but not C6 glioma cells or neuronal cultures (no glia) from the mesencephalon. The pure neuronal cultures were destroyed by incubation withl‐DOPA; added ascorbic acid or superoxide dismutase protected the cells. Washout ofl‐DOPA after 48 h amplified the rise in GSH content in mixed cultures (neurons plus glia). Examination of structure‐activity relationships for elevating GSH levels in responsive cell types revealed that autooxidizable compounds (α‐methyl‐DOPA, dopamine, apomorphine, catechol, and hydroquinone) behaved similarly tol‐DOPA, whereas structural analogues that cannot undergo autooxidation (3‐O‐methyl‐DOPA, tyrosine, 2,4‐dihydroxyphenylalanine, and resorcinol) failed to elevate GSH levels. Therefore, up‐regulation of GSH appears to be a response to a mild oxidative stress. When mixed mesencephalic cultures were exposed to a strong oxidant stress by incubation withtert‐butyl hydroperoxide, a loss in viability was seen. Cultures pretreated withl‐DOPA or hydroquinone were protected from loss of viability. However, when cultures were pretreated with bothl‐DOPA and ascorbate, which prevents the rise in GSH level, protection was lost, in accord with the failure to up‐regulate GSH. These results show that the up‐regulation of cellular GSH evoked by autooxidizable agents is associated with significant protection of cells. Glia play an essential role in the response of mesencephalic cell cultures. An ability to up‐regu

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66020501.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We have previously reported that insulin/insulin‐like growth factor (IGF)‐I induced the α1 isoform of Na+,K+‐ATPase in cultured astrocytes. In this study the effects of insulin/IGF‐I on Na+,K+‐ATPase activity and cell proliferation were examined in astrocytes cultured under the various conditions, to test the possible involvement of the enzyme activity in the mitogenic action of IGF‐I on astrocytes. Insulin increased Na+,K+‐ATPase activity and stimulated cell proliferation in subconfluent astrocytes (cultured for 7–14 days in vitro). In contrast, these effects were not observed in confluent cells (cultured for 28 days). Furthermore, insulin stimulated neither the enzyme activity nor [3H]thymidine incorporation in astrocytes preincubated in fetal calf serum‐free medium for 2 days (quiescent cells) and treated with dibutyryl cyclic AMP (differentiated cells). The increases in Na+,K+‐ATPase activity and expression of the α1 mRNA preceded the mitogenic effect.125I‐IGF‐I binding experiment showed that all the cells used here had similar binding characteristics. The insulin‐induced increase in enzyme activity was not affected by 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H‐7), and it was observed even in Ca2+‐free medium. The stimulation by IGF‐I of [3H]thymidine incorporation was attenuated by ouabain and a low external K+level. These findings suggest that stimulation of Na+,K+‐ATPase activity is involved in the mitog

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66020511.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The effect of lithium on inositol phospholipid resynthesis in primary cultures of cerebellar granule cells was studied. During activation of phospholipase C by the combined action of a muscarinic agonist and mild depolarization, the levels of inositol phospholipids as well as the inositol phospholipid precursor CMP‐phosphatidate appeared highly sensitive to lithium with half‐maximal accumulation of CMP‐phosphatidate attained at 0.5 mMLiCl, a concentration close to that in the plasma of patients subjected to lithium therapy. Under the same conditions, the effect of lithium on inositol phospholipid metabolism appeared to be mediated by depletion of cytoplasmic free inositol content. This was indicated by the observation that preincubation for 48 h in high extracellular inositol concentrations could decrease or delay the depletion of inositol phospholipids and the accumulation of CMP‐phosphatidate induced by 10 mMLiCl. Because even relatively high concentrations of extracellular inositol (500 µM) only partially prevented inositol phospholipid depletion, cerebellar granule cells appear to have a comparatively low capacity to accumulate inositol intracellularly, in comparison with other brain cells in culture. The relationship between CMP‐phosphatidate accumulation and phospholipase C activity has also been investigated using a range of agonists that have been reported to act on cerebellar gra

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66020517.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY