摘要:

Abstract:Previous reports have demonstrated that glutamate stimulates c‐fosmRNA expression in primary cultures of mouse cerebral cortical neurons. We show here that vasoactive intestinal peptide (VIP) induces c‐fosmRNA expression; however, this effect of VIP is completely inhibited by the noncompetitive NMDA receptor antagonist MK‐801, therefore indicating that VIP stimulates c‐fosexpression in a glutamate‐dependent manner. A similar effect was observed with pituitary adenylate cyclase‐activating polypeptide27 (PACAP27). At the intracellular level, coactivation of protein kinases A and C mediates the glutamate‐dependent stimulation of c‐fosexpression evoked by VIP, because either H‐89 or staurosporin inhibits the effect of VIP as well as that of glutamate. These results point to a “biochemical AND gate” mechanism, which implies the obligatory activation of both protein kinases A and C in the transduction of c‐fosexpression. In summary, this article provides evidence that VIP and PACAP27 potentiate the effect of glutamate, the principal effector on c‐fosexpression, suggesting that both peptides can increase the “throughput” or “strength” of glutamate‐contain

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65010001.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We have isolated several new genes that are specifically expressed by oligodendrocytes in the CNS. This was achieved by differential screening of a rat spinal cord cDNA library with probes derived from normal and from oligodendrocyte‐free spinal cord mRNAs. Four of these genes are exclusively expressed by oligodendrocytes: Three of these are not related to known genes, whereas one encodes the myelin oligodendrocyte glycoprotein (MOG). Four other genes are expressed by oligodendrocytes as well as by Schwann cells. One gene codes for apolipoprotein D, which is thought to be involved in lipid metabolism. A second cDNA sequence codes for the recently identified galactosylceramide‐synthesizing enzyme UDP‐galactose:ceramide galactosyl‐transferase. The third gene encodes a small protein with four putative transmembrane domains that is related to a T‐lymphocyte‐specific membrane protein, MAL. The fourth gene encodes the rat homologue of the stearyl‐CoA‐desaturase 2 (SCD2) gene, which is specifically expressed in the nervous system and involved in the synthesis and regulation of long‐chain unsaturated fatty acids essential for myelination. Finally, we found that a member of the β‐tubulin family is highly expressed in oligodendrocytes as well as neurons. The identification of several new proteins that may play a role in myelin synthesis and sheath formation will lead to new insight into th

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65010010.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Cell type‐specific expression of the catecholamine synthetic enzyme, tyrosine hydroxylase (TH), appears to be mediated in part bycis‐acting elements located at the 3′ end of the human gene. Further delineation of this region indicated sequences corresponding to a CACGTG motif significantly stimulated transcription of a heterologous promoter in various cell types. Mutation of this site led to a complete loss of activity. DNase footprinting, gel retardation, and UV cross‐linking experiments indicated that a 74‐kDa cellular factor(s) bound specifically to the CACGTG motif in the pheochromocytoma cell line PC12. The size of this protein and its pattern of expression are compatible with those of the CACGTG binding protein TFE3. Transgenic animals were created using a 261‐bp human TH 3′ fragment encompassing the CACGTG motif in front of a thymidine kinase promoter/chloramphenicol acetyltransferase reporter gene. In three lines of mice this fragment was sufficient to direct a pattern of mRNA expression in peripheral neuroendocrine tissues that mimicked TH mRNA distribution. However, these sequences were not sufficient for CNS‐specific patterns of expression. Thus, multiple cell type‐specific enhancers may regulate TH gene expression in the

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65010023.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Synaptotagmin is a synaptic vesicle specific protein that binds calcium and phospholipids in vitro and is required for calcium‐regulated fusion of synaptic vesicles with the presynaptic membrane. We have examined the possible requirement for synaptotagmin in axonal outgrowth by following neuronal development inDrosophilaembryos deficient for the synaptotagmin gene. We find that synaptotagmin is expressed abundantly in axons and growth cones before synapse formation in wild‐type embryos. Using antibodies to the intravesicular domain of synaptotagmin to label live embryos, we demonstrate that vesicle populations containing synaptotagmin actively undergo exocytosis during axonogenesis. We have used immunocytochemical techniques to examine the distribution of the axonal protein Fasciclin II, the presynaptic membrane protein syntaxin, and the synaptic vesicle protein cysteine string protein, in synaptotagmin null mutations. The distribution of these proteins is similar in wild‐type and synaptotagmin mutant embryos, suggesting that synaptotagmin is not required for axonogenesis in the CNS or PNS. Based on these findings, we suggest that the molecular mechanisms underlying vesicular‐mediated membrane expansion during axonal outgrowth are distinct from those required for synaptic vesicle fusion during neurotransmitter

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65010032.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Regulation of the initial rate of uptake and steady‐state concentration of ascorbate (reduced vitamin C) was investigated in rat cerebral astrocytes. Although these cells did not synthesize vitamin C, they accumulated millimolar concentrations of ascorbate when incubated with medium containing the vitamin at a level (200 µM) typical of brain extracellular fluid. Initial rate of [14C]‐ascorbate uptake and intracellular ascorbate concentration were dependent on extracellular Na+and sensitive to the anion transport inhibitor sulfinpyrazone. Comparison of the efflux profiles of ascorbate and 2′,7′‐bis(carboxyethyl)‐5 (or ‐6)‐carboxyfluorescein from astrocytes permeabilized with digitonin localized most intracellular ascorbate to the cytosol. Pretreatment of astrocytes with dibutyryl cyclic AMP (dBcAMP) doubled their initial rate of sulfinpyrazone‐sensitive [14C]ascorbate uptake compared with cells treated with eithern‐butyric acid or vehicle. dBcAMP also increased steady‐state intracellular ascorbate concentration by 39%. The relatively small size of the change in astrocytic ascorbate concentration was explained by the finding that dBcAMP increased the rate of efflux of the vitamin from ascorbate‐loaded cells. These results indicate that uptake and efflux pathways are stimulated by cyclic AMP‐dependent mechanisms and that they regulate the cytosolic concentration

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65010041.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We investigated the effects of retinoids on the cholinergic properties of a murine septal cell line, SN56. Treatment of the cells with all‐trans‐retinol (vitamin A), all‐trans‐retinal, all‐trans‐retinoic acid (t‐RA), 9‐cis‐retinoic acid (9c‐RA), or 13‐cis‐retinoic acid caused time‐ and concentration‐dependent increases in choline acetyltransferase activity (up to 3.4‐fold) and in intracellular acetylcholine levels (up to 2.5‐fold, with respective EC50values of 68, 50, 18, 15, and 56 nM). Furthermore, treatment with either t‐RA or 9c‐RA at 1 µMfor 48 h resulted in an increase in the expression of choline acetyltransferase mRNA by threefold that of controls. These data and the presence of putative retinoic acid response elements in the 5′ region of the murine choline acetyltransferase gene indicate that retinoids stimulate choline acetyltransferase transcription in murine cholinergic neurons. No additivity or synergism was observed between the effects of t‐RA and 9c‐RA on any of these cholinergic properties of SN56 cells, suggesting a common mechanism of action of the two retinoids. However, a combined treatment with t‐RA and forskolin, which activates adenylate cyclase, resulted in an additive increase in acetylcholine content. Using an antagonist selective for the retinoic acid receptor‐α subtype, Ro 41‐5253, we found that the effects of t‐RA and 9c‐RA on acetylcholine levels were abolished. An agonist selective for retinoic acid receptor‐α, Ro 40‐6055, increased acetylcholine levels to a similar extent as t‐RA and 9c‐RA, and this effect was blocked by the antagonist. Our results suggest that retinoids modulate the cholinergic phenoty

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65010050.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Evidence from in vitro studies suggests that excitotoxic neuronal degeneration can occur by either an acute or delayed mechanism. Studies of the acute mechanism in isolated chick embryo retina using histological methods indicate that this process is rapidly triggered by activation of glutamate receptors of either theN‐methyl‐d‐aspartate (NMDA) or non‐NMDA subtypes. The delayed mechanism, studied primarily in cortical and hippocampal cell cultures prepared from embryonic rodent brain, requires activation of NMDA receptors. In these cell culture systems, stimulation of non‐NMDA receptors does not rapidly trigger delayed neuronal degeneration, or does so only indirectly, via activation of NMDA receptors secondary to glutamate release. To provide a more valid basis for comparison of these two mechanisms, we have modified the isolated chick embryo retina model to permit studies of delayed as well as acute excitotoxic neurodegeneration. Retinas maintained for 24 h exhibited no morphological or biochemical signs of damage. Retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium at various times after exposure to agonists and normalized to total LDH in each retina. Glutamate exposure (1 mM, 30 min) did not result in LDH release by the end of the exposure period, but LDH was released over the following 24 h. Briefer periods also led to substantial LDH release. Incubation in the presence of NMDA, or the non‐NMDA agonists kainate (KA) or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), led rapidly to delayed LDH release. NMDA and AMPA were more potent than glutamate, but high concentrations of glutamate led to more LDH release than high concentrations of these agonists. KA was a powerful excitotoxin, providing more LDH release than glutamate, NMDA, or AMPA at every concentration tested. The delayed LDH release induced by glutamate involved activation of both NMDA and non‐NMDA receptors, as a combination of receptor‐selective antagonists was necessary to provide complete blockade. These results indicate that glutamate, NMDA, AMPA, and KA all cause delayed as well as acute excitotoxic damage in the retina. It is interesting that brief exposure to the non‐NMDA receptor agonists, in relatively low concentrations, led to delayed LDH release. This is different than in other in vitro models of delay

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65010059.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Recent reports suggest that nitric oxide (NO) may contribute to several neurodegenerative diseases, e.g., focal cerebral ischemia,N‐methyl‐d‐aspartate‐mediated neurotoxicity, and experimental autoimmune encephalomyelitis. Accordingly, an understanding of the CNS transport processes of NO synthase (NOS) inhibitors has important therapeutic implications. The objective of the present study was to characterize the in vitro transport processes governing the uptake ofl‐[14C]arginine and the NOS inhibitor [14C]aminoguanidine in rat choroid plexus tissue. Consistent with previous reports, the uptake ofl‐[14C]arginine was mediated by both saturable and nonsaturable processes and was inhibited by the NOS inhibitorsNG‐methyl‐l‐arginine,NG‐amino‐l‐arginine, andN5‐imidoethyl‐l‐ornithine.l‐[14C]Arginine uptake was not inhibited by aminoguanidine orNG‐nitro‐l‐arginine. Because aminoguanidine is an organic cation that bears some structural similarity tol‐arginine, aminoguanidine might be transported by either an organic cation transporter or by the basic amino acid transporter governing arginine uptake. However, there was no evidence of a saturable uptake process for [14C]aminoguanidine in isolated rat choroid plexus, in contr

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65010068.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:In primary cultures of mouse cerebellar granule cells, a brief stimulation by γ‐hydroxybutyric acid (GHB, 0.1–3 mM) significantly increased the intracellular Ca2+concentration ([Ca2+]i) in a concentration‐dependent manner. In addition, gel mobility assay showed that exposure of the cells to GHB also increased nuclear DNA‐binding activity specific for the cyclic AMP‐responsive element (CRE) and activator protein 1 (AP‐1) transcriptional element in a concentration‐dependent manner. The concentration range of GHB that increased the DNA‐binding activity was essentially the same as the concentration range that elicited the increase in [Ca2+]i. The GHB‐induced increases in [Ca2+]iand nuclear DNA‐binding activity were antagonized by specific GABABantagonists such asp‐[3‐aminopropyl]‐p‐diethoxymethylphosphinic acid (CGP 35 348) and 3‐N‐[1‐(S)‐(3,4‐dichlorobenzyl)ethanol‐2‐(S)‐hydroxy‐P‐benzylphosphinic acid (CGP 55 845). In addition, the GHB‐induced increase in [Ca2+]iwas abolished by pretreatment of the cells with islet‐activating protein. Furthermore, treatment of the cells with 1,2‐bis(2′‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetraacetoxymethyl ester (BAPTA‐AM) and thapsigargin blocked the GHB‐induced increase in nuclear DNA‐binding activity. GHB inhibited [3H]baclofen binding to cultured cerebellar granule cells and mouse cerebellar membranes. These results suggest that stimulation of GABABreceptors by GHB activates intracellular Ca2+stores and that the increased [Ca2+]iresulting from release of stored Ca2+plays an important role in increasing the C

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65010075.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Triggering of the cell adhesion molecules L1 or N‐CAM in a nerve growth cone membrane fraction from fetal rat brain with purified L1 or N‐CAM or specific antibodies decreases the steady‐state levels of protein tyrosine phosphorylation in the membranes. Here we report that triggering of L1 and N‐CAM in the growth cone‐enriched membrane fraction with a subset of antibodies directed against the extracellular region of L1 and N‐CAM elicited dephosphorylation of endogenous protein substrates, indicating the presence of a cell adhesion molecule‐activated phosphatase. The most prominent substrates were a membrane‐associated 200‐kDa protein and tubulin, both of which were dephosphorylated on tyrosine and serine/threonine residues in response to L1 or N‐CAM triggering. The antibody‐induced phosphatase was inhibited by agents that blocked tyrosine and serine/threonine phosphatases, including sodium orthovanadate, vanadyl sulfate, zinc cations, heparin, and sodium pyrophosphate. Purified L1 and N‐CAM fragments and other antibodies reacting with the extracellular region of these adhesion molecules did not activate the phosphatase but did inhibit tyrosine phosphorylation. These properties suggested that triggering of L1 and N‐CAM can lead to either phosphatase activation or tyrosine kinase inhibition in growth cone membranes. These findings implicate protein phosphatases in addition to tyrosine kinases as components of L1 and N‐CAM intracellular signali

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65010084.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY