首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 0 毫秒
1.
Abstract: R -Deprenyl and R -2-heptyl- N -methylpropargylamine ( R -2-HMP) are compounds that have been shown to reduce neuronal death in various in vitro and in vivo models involving apoptosis but do not always prevent apoptosis. In the present study we have examined the effects of these compounds and their S enantiomers on cytosine arabinoside (ara C)-induced apoptosis and low K+-induced apoptosis in cerebellar granule cells in primary culture. It was found that R -deprenyl and R -2-HMP could prevent ara C-induced apoptosis with an EC50 around 10−9 M but could not prevent low K+-induced apoptosis. S -Deprenyl and S -2-HMP did not prevent apoptosis under any conditions but were found to antagonize the antiapoptotic actions of R -deprenyl and R -2-HMP. Using the fluorescent mitochondrial dye chloromethyltetramethylrhodamine methyl ester it was found that there was a loss of mitochondrial function in cerebellar granule cells exposed to ara C but not low K+ medium. R -Deprenyl and R -2-HMP prevented the ara C-induced loss of mitochondrial function. It is concluded that R -deprenyl and R -2-HMP prevent apoptosis of cerebellar granule cells by a mechanism that is independent of monoamine oxidase inhibition and that they act on the same site to prevent specifically apoptosis involving a loss of mitochondrial membrane potential, possibly p53-dependent apoptosis.  相似文献   

2.
3.
Abstract: The effect(s) of a prototypic intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca2+ levels ([Ca2+]i) that was dependent on the extracellular concentration of Ca2+ ([Ca2+]o). In addition, this increase in [Ca2+]i correlated with a decrease in cell viability that was also dependent on [Ca2+]o. Glutamate-induced toxicity, quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an “early” Na+/Cl?-dependent component observed within minutes of glutamate exposure, and a “delayed” Ca2+-dependent component (ED50~50 µM) that coincided with progressive degeneration of granule cells 4–24 h after a brief (5–15 min) exposure to 100 µM glutamate. Quantitative analysis of cell viability and morphological observations identify a “window” in which TMB-8 (at >100 µM) protects granule cells from the Ca2+-dependent, but not the Na+/Cl?-dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca2+]i. These findings suggest that Ca2+ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca2+]i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca2+ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca2+]i and glutamate-induced neurotoxicity.  相似文献   

4.
The regulation of the cytosolic free Ca2+ concentration ([Ca2+]i) was investigated by microfluorimetry in single cerebellar granule neurons exposed to various treatments (high K+, glutamate, or acetylcholine) and drugs. The responses to the treatments developed asynchronously during cell culture, with high K+ and glutamate reaching their maxima at 6 and 7 days in vitro and acetylcholine at 9 days in vitro. The biphasic [Ca2+]i transients induced by high K+ (an initial peak, followed by a plateau 30-40% of the peak, both sustained by dihydropyridine-sensitive voltage-gated Ca2+ channels) were dissipated by washing with fresh medium or, more rapidly, by addition of excess EGTA (t1/2 = 11 +/- 2 and 3 +/- 0.6 s, respectively). Compared to those induced by high K+, the [Ca2+]i transients induced by glutamate administered in Mg2(+)-free medium were much more variable. An initial peak, sustained by voltage-gated Ca2+ channels, was visible in only approximately 50% of the cells and disappeared when multiple glutamate pulses were administered. In the rest of the population, the transients were monophasic, with persistent plateaus sustained only in part (30-40%) by voltage-gated Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
Abstract: We analyzed biochemically and temporally the molecular events that occur in the programmed cell death of mouse cerebellar granule neurons deprived of high potassium levels. An hour after switching the neurons to a low extracellular K+ concentration ([K+]o), a significant part of the genomic DNA was already cleaved to high-molecular-weight fragments. This phenomenon was intensified with the progression of the death process. Addition of cycloheximide to the neurons 4 h after high [K+]o deprivation resulted in no cell loss and complete recovery of the damaged DNA. DNA margination and nuclear fragmentation as assessed by 4,6-diaminodiphenyl-2-phenylindole staining were observable in a few cells beginning ~4 h after the removal of high [K+]o and developed to nuclear condensation 4 h later. Six hours after high [K+]o deprivation, the DNA was fragmented into oligonucleosome-sized fragments. Within 6 h after removal of the extracellular K+, 50% of the neurons were committed to die and lost their ability to be rescued by readministration of 25 mM [K+]o. Similar to high [K+]o deprivation, inhibition of RNA or protein synthesis failed to halt neuronal degeneration of a similar percentage of cells 6 h after the onset of the death process. Mitochondrial function steadily decreased after [K+]o removal. An ~40% decrease in RNA and protein synthesis was detected by 6 h of [K+]o removal during the period of cell death commitment; rates continued to decline gradually thereafter. The temporal characteristics of the DNA damage and recovery, DNA cleavage to oligonucleosome-sized fragments, and the reduction in mitochondrial activity—events that occurred within the critical time—may indicate that these processes have an important part in the mechanism that committed the neurons to die.  相似文献   

6.
We compared neurotoxicity of piperine and low K+on cultured cerebellar granule neurons. As considered from lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide reduction, both piperine and shifting from high K+(25 mM) to low K+(5.4 mM) were cytotoxic to cerebellar granule neurons. Protein synthesis inhibitors, cycloheximide and anisomycin, and an endonuclease inhibitor, aurintricarboxylic acid, were protective against low K+-induced neuronal death whereas they were ineffective against that induced by piperine. D--tocopherol, trolox, and a spin trap 3,3,5,5-tetramethyl-l-pyrroline-l-oxide were protective against piperine neurotoxicity whereas they had no effect on that induced by low K+. These results suggest that piperine and low K+may exert neurotoxic effects on cerebellar granule neurons through different mechanisms. Death of cerebellar granule neurons induced by piperine may be mediated by non-apoptotic mechanisms and may involve membrane lipid peroxidation and/or free radical generation.  相似文献   

7.
Abstract: Excitatory amino acid (EAA)-induced polyphosphoinositide (PPI) hydrolysis was studied during the development in culture of cerebellar granule cells. The developmental pattern was similar using metabotropic glutamate (Glu) receptor (mGluR) agonists, including L-Glu, quisqualate, and trans -(±)-1-amino-1,3-cyclopentanedicarboxylic acid: The stimulation of [3H]inositol monophosphate ([3H]-InsP) formation was low at 2 days in vitro (DIV), but the response increased steeply, reaching a peak at 4 DIV, followed by a progressive decline. In contrast, carbamylcholine-induced PPI hydrolysis exhibited a plateau after a pronounced increase during the first week in vitro. At 6 DIV, but not at 4 DIV, when the activity peaked, PPI hydrolysis elicited by Glu was reduced by the N -methyl- d -aspartate (NMDA) receptor antagonist MK-801, indicating that in cultured granule cells, NMDA receptors contribute to [3H]-InsP formation and that this component of the response develops relatively late. Accordingly, NMDA-induced [3H]-InsP formation, estimated under Mg2+-free conditions, increased markedly from very low values at 2 DIV to a plateau at 8–10 DIV. The developmental pattern of EAA-induced PPI hydrolysis was paralleled by changes in the level of an mRNA for a specific mGluR subtype ( mGluR1 mRNA). RNA blot analysis performed with the pmGR1 cDNA probe revealed that the hybridization signal in RNA extracts from cultures at 1 DIV was very weak, but mGluR mRNA levels increased dramatically between 1 and 3 DIV, followed by a progressive decrease, so that by 15 DIV the mRNA levels were only ∼10% of the values at 3 DIV. These observations indicate that the functional expression of the mGluR is subject to developmental regulation, which critically involves receptor mRNA levels.  相似文献   

8.
Abstract: Pigment epithelium-derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y-79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose-response relationship, with a larger effect in chemically defined medium than in serum-containing medium [ED50 = 30 ng/ml (0.70 n M ) in chemically defined medium and 100 ng/ml (2.3 n M ) in serum-containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme-linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.  相似文献   

9.
Abstract: Activation of the N-methyl-d -aspartate (NMDA) subtype of glutamate receptor increases levels of intracellular calcium and can lead to stimulation of protein kinase C activity. Several reports have demonstrated that stimulation of protein kinase C can, in turn, increase electrophysiological responses to NMDA in certain cells or in oocytes expressing certain NMDA receptor subunits. In the present study, the effects of protein kinase C activation on NMDA receptor-mediated increases in intracellular Ca2+ levels were investigated in primary cultures of rat cerebellar granule cells using fura-2 fluorescence spectroscopy. Pretreatment of the cells with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), but not the inactive analogue 4α-phorbol 12-myristate 13-acetate, inhibited NMDA-induced increases in intracellular Ca2+ levels. Coincubation of cells with PMA and the kinase inhibitor staurosporine or calphostin C blocked the PMA effect. The potency of NMDA was reduced twofold, and the potency of the NMDA receptor coagonist, glycine, to enhance the response to NMDA was decreased fourfold by pretreatment of cells with PMA. The effect on glycine was mimicked by pretreatment with okadaic acid, a protein phosphatase inhibitor. PMA treatment did not significantly alter Mg2+ inhibition of the NMDA response but decreased the potency of the competitive antagonist CGS-19755. These data suggest that, in cerebellar granule cells, the function of the NMDA receptor may be subject to feedback inhibition by protein kinase C stimulation. Under physiological conditions, this inhibition may result from a decreased effectiveness of the endogenous coagonists, glutamate and glycine.  相似文献   

10.
Maitotoxin (MTX) stimulated inositol phosphate (IP) formation in primary cultures of rat cerebellar granule cells. MTX-induced IP production was dependent on extracellular Ca2+ but independent of extracellular Na+. The stimulation of IP formation elicited by MTX was unaffected by pretreatment of cells with phorbol dibutyrate, pertussis toxin, and a variety of Ca2+ entry blockers, such as nimodipine, nisoldipine, Co2+, and Mn2+. The presence of MTX markedly attenuated IP production induced by carbachol and glutamate, with no apparent effect on the responses to norepinephrine (NE), histamine, 5-hydroxytryptamine (5-HT), and endothelin-1. The inhibition of the carbachol- and glutamate-induced responses by MTX was dose dependent with IC50 values of 1.2 and 0.5 ng/ml, respectively. Pretreatment of cells with a lower concentration of MTX (0.3 ng/ml) also attenuated carbachol- and glutamate-induced IP formation, in a time-dependent manner, with a decrease observed after 30 min prestimulation, but failed to affect NE-, histamine-, 5-HT-, endothelin-1, and sarafotoxin S6b-induced responses. Thus, MTX elicited a marked Ca2(+)-dependent phosphoinositide (PI) turnover in cerebellar granule cells and selectively inhibited carbachol- and glutamate-induced PI hydrolysis. Possible mechanisms underlying these selective modulations are discussed.  相似文献   

11.
Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca2+ medium; whereas in the presence of Ca2+, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca2+ ([Ca2+]c). The plateau component of the increase can be inhibited additively by the L-type Ca2+ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca2+]c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca2+ dependent for the first 6–10 min after the evoked increase in [Ca2+]c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.  相似文献   

12.
Abstract: When primary cultures of cerebellar granule neurons are grown in a physiological concentration of KCl (5 m M ) they undergo apoptosis, which can be prevented by growing the cells in the presence of N -methyl- d -aspartate (NMDA). We now show that ethanol inhibits this trophic effect of NMDA, i.e., promotes apoptosis, and also inhibits the NMDA-induced increase in intracellular Ca2+ concentration in cells grown in 5 m M KCl. Both effects of ethanol show a similar concentration dependence and are reversed by a high concentration of glycine, the co-agonist at the NMDA receptor. The data suggest that the effect of ethanol on apoptosis is mediated, at least in part, by inhibition of NMDA receptor function. This effect of ethanol to increase apoptosis could contribute to the previously described in vivo sensitivity of the developing cerebellum to ethanol-induced damage.  相似文献   

13.
The regulation and expression of muscarinic acetylcholine receptor (mAChR) mRNA was studied in cultured cerebellar granule cells using Northern blot hybridization, mRNA species for m2- and m3-mAChRs but not m1- and m4-mAChRs were detected in these cells. The expression of mRNAs of both m2- and m3-mAChRs reached a maximum on the tenth day in culture but their expression patterns differed. Treatment of cerebellar granule cells after 8 days in culture with 100 microM carbachol led to differential down-regulation of the mRNA species of both mAChR subtypes present. Muscarinic receptor antagonists, atropine (1 microM) and pirenzepine (10 microM), prevented carbachol-induced m3-mAChR mRNA down-regulation observed at 8 h. However, exposure to either atropine or pirenzepine alone for 8 h led to a significant up-regulation of m3-mAChR mRNA. Thus, the mRNA species for both m2- and m3-mAChR subtypes are differentially expressed in culture and down-regulated by agonist stimulation. The loss of these mRNA species may play a role in the down-regulation of mAChR binding sites that occurs after desensitization.  相似文献   

14.
15.
Abstract: The ability of ethanol to interfere with insulin-like growth factor 1 (IGF-1)-mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 m M K+ to one containing 5 m M K+. IGF-1 protected granule neurons from apoptosis in medium containing 5 m M K+. Ethanol inhibited IGF-1-mediated neuronal survival but did not inhibit IGF-1 receptor binding or the neurotrophic action of elevated K+, and failed to potentiate cell death in the presence of 5 m M K+. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF-1. Significant inhibition by ethanol (15–20%) was observed at 1 m M and was half-maximal at 45 m M . The inhibition of IGF-1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3-kinase (PI 3-kinase), and a block of IGF-1-stimulated PI 3-kinase activity. The neurotrophic response of IGF-1 was also inhibited by the PI 3-kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF-1.  相似文献   

16.
By use of nuclear mini-extracts prepared from cultured cerebellar granule cells in a gel-mobility assay, exogenous N-methyl-D-aspartate (NMDA) or kainate was shown to increase both 12-O-tetradecanoylphorbol 13-acetate-responsive element (TRE)- and cyclic AMP-responsive element (CRE)-binding activity. These increases were specifically prevented by the NMDA receptor antagonist D,L-2-amino-5-phosphonovalerate and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. The increase of TRE-binding activity was dependent on de novo protein synthesis, and its inductions by both NMDA and kainate required extracellular Ca2+. TRE-binding activity was competitively inhibited by the CRE, and vice versa, showing higher DNA-binding affinity to the CRE than to the TRE. A proteolytic clipping bandshift assay demonstrated that the increase in CRE-binding activity could be mediated by the TRE-binding activity. Thus, the TRE-binding activity cross-binding to the CRE could be activated by NMDA or kainate stimulation. The involvement of c-Fos or Fos-related proteins in the TRE- and CRE-binding complexes was shown by a supershift gel-mobility assay using anti-c-Fos antiserum.  相似文献   

17.
Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.  相似文献   

18.
Abstract: Tryptamine dose-dependently increased phosphoinositide (PI) hydrolysis by approximately fourfold in primary cultures of rat cerebellar granule cells (EC50 = 56 µ M ). The PI response stimulated by tryptamine was dependent on the presence of extracellular Ca2+ and Na+. Tryptamine-induced PI breakdown could be partially inhibited by pretreatment with 4β-phorbol 12-myristate 13-acetate but not pertussis toxin. The presence of tryptamine markedly attenuated PI responses induced by norepinephrine (NE) and carbachol, with no apparent effect on the responses to 5-hydroxytryptamine and glutamate. The inhibition of NE- and carbachol-induced PI turnover by tryptamine was dose dependent with IC50 values of ∼0.4 and ∼2.5 m M , respectively. Pretreatment of cells with tryptamine (0.5 m M ) also attenuated NE- and carbachol-induced PI turnover, but failed to affect 5-hydroxytryptamine- and glutamate-induced responses. Furthermore, ketanserin, atropine, and prazosin did not have any effect on inositol phosphate formation induced by tryptamine. These observations indicate that tryptamine markedly increased Ca2+- and Na+-dependent PI turnover in cerebellar neurons and selectively inhibited NE- and carbachol-induced PI hydrolysis.  相似文献   

19.
Abstract: The [Ca2+]1 of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K+ level), which both activate Ca2+ influx. The possibility that these stimuli activate Ca2+-induced Ca2+ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca2+]1 response to K+ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca2+], response to K+ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K+-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K+. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K+- and NMDA-induced elevation of [Ca2+]1 appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca2+ pools.  相似文献   

20.
S1P is involved in the regulation of multiple biological processes (cell survival, growth, migration and differentiation) both in neurons and glial cells. The study was aimed at investigating the possible effects of S1P on calcium signaling in cerebellar astrocytes and differentiated granule cells. In cerebellar astrocytes S1P is able to mediate calcium signaling mainly through Gi protein coupled receptors, whereas in differentiated neurons it failed to evoke any calcium signaling, despite acting both extracellularly and intracellularly. The data indicate strict cell specificity in S1P-evoked calcium response, which could be relevant to communication between neurons and glial cells in the cerebellum.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号