摘要:

Abstract:The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92‐kDa component isolated from a detergent‐insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full‐length cDNA, encoding the 92‐kDa component, using both cDNA library screening and 5′‐rapid amplification of cDNA ends (5′‐RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of theDrosophila trpandtrplgenes. Greatest variation from theDrosophilaTrp protein is seen in the carboxyl‐terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl‐terminal domain has been shown to be in the regulation of several ligand‐gated channels. The carboxyl‐terminal domain has been expressed and shown to interact with calmodulin in a calcium‐dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels invol

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67062227.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Acetylcholine and other muscarinic agonists stimulate the proliferation of rat cortical astrocytes and 132 1N1 human astrocytoma cells by activating muscarinic m3cholinergic receptors. Ethanol was a potent inhibitor of carbachol‐stimulated proliferation, measured by [3H]thymidine incorporation, with an IC50of 10 mM. On the other hand, basal and serum‐stimulated proliferation of astrocytes and astrocytoma cells was inhibited by ethanol with lower potency (IC50= 200–250 mM). Concentration‐response experiments with carbachol, in the presence of 10 mMethanol, suggested that inhibition of proliferation by the alcohol was of the noncompetitive type. Experiments with acetaldehyde and with the alcohol dehydrogenase inhibitor 4‐methylpyrazole suggested that the inhibitory effect of alcohol was due to ethanol itself and not to its metabolite acetaldehyde. Proliferation of astrocytoma cells induced by carbachol and the inhibitory effects of ethanol were also confirmed by flow cytometry using the 5‐bromodeoxyuridine‐Hoechst 33258 method. Ethanol (10 mM) had no effect on proliferation induced by 50 µg/ml insulin and 100 ng/ml platelet‐derived growth factor BB; on the other hand, the mitogenic effect of 1 mMhistamine, 100 U/ml interleukin‐1, and 100 ng/ml 12‐O‐tetradecanoylphorbol 13‐acetate were inhibited by ∼50%. These results indicate that proliferation of glial cells induced by muscarinic agonists is especially sensitive to the inhibitory effect of ethanol. This action of ethanol may be relevant to its developmental neurotoxicity, particularly microencephaly, which is one of the common features of

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67062236.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The cyclic AMP (cAMP)‐induced inhibitory effect on cell proliferation was examined through inhibition of mitogen‐activated protein kinase (MAP kinase) activation in cultured rat cortical astrocytes. Basic fibroblast growth factor (bFGF) at 10 ng/ml maximally stimulated MAP kinase activity, which peaks during 10 min and prolonged for 24 h. Likewise, DNA synthesis was maximally potentiated with 10 ng/ml bFGF and correlated with MAP kinase activity in a dose‐dependent manner. Dibutyryl cAMP (dbcAMP) at 1 mMand isoproterenol at 10 µMinhibited MAP kinase activation and DNA synthesis potentiation with bFGF and platelet‐derived growth factor to the control level in cultured astrocytes and C6 glioma cells. The stimulation with bFGF caused a prominent translocation of MAP kinase from the cytosol to the nucleus after 1 h in astrocytes. Treatment of the cells with dbcAMP and isoproterenol completely prevented the translocation of MAP kinase. In experiments with32P‐labeled cultured astrocytes, phosphorylation of Raf‐1 was apparently stimulated with bFGF. Treatment with dbcAMP or isoproterenol had a greatly inhibitory effect on the stimulation of Raf‐1 phosphorylation with bFGF. Consistent with the effect on Raf‐1 phosphorylation, dbcAMP and isoproterenol completely prevented bFGF‐induced phosphorylation of MAP kinase kinases, target proteins of Raf‐1. Our observations suggest that cAMP‐induced suppression of cell growth in astrocytes is due to the inhibitory effect on activation of MAP kinase and its translocation to the nucleus and that the site of the cAMP action is located at Raf‐1 or th

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67062246.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Chromaffin cells of the adrenal medulla are neural crest‐derived neuroendocrine cells that express neuropeptide genes in vivo and in vitro. As such these cells are useful for examining tissue‐ and cell‐specific regulation of the enkephalin gene. We previously demonstrated that the chromatin configuration of the enkephalin gene correlated with its tissue‐specific expression in the adrenal medulla and primary chromaffin cell cultures. In this study we examine and characterize binding of transcription factors to the enkephalin promoter/enhancer region. Gel shift analyses of this region with extracts from chromaffin cells and PC12 cells (a pheochromocytoma cell line that does not express the enkephalin gene) demonstrate that all detectable binding is to ENKCRE‐2, a cyclic AMP response‐like element, and that the binding is cell specific. Gel shift and supershift analyses show that, unlike reports demonstrating that binding activity in the CNS is composed of the cyclic AMP response element binding protein, CREB, the majority of protein binding in chromaffin cells is from the AP‐1 family of transcription factors. This binding is composed of c‐Jun, JunD, and possibly a novel Fos‐related protein(s). These data suggest enkephalin gene expression in the adrenal gland is controlled by cell‐specific binding of transcription factors from the Fos/Jun families to the enkephalin CRE‐2 element. Furthermore, these data suggest at least two different modes of enkephalin gene regulation exist between endocrine

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67062256.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Little is known about the role of theN‐linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF‐R). In a human glioma cell line, U373 MG, EGF‐Rs contain the bisectingN‐linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin fromPhaseolus vulgaris(E‐PHA). Incubation of E‐PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF‐induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF‐R, phenotypic events that depend on EGF‐R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin fromP. vulgaris(L‐PHA), an isolectin of E‐PHA, had no effect on EGF‐R activity or the biological functions of these cells even though L‐PHA was able to bind to the EGF‐R. These findings suggest the presence of an important bisectingN‐linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E‐PHA lectin binding may provide an additional approach to blocking E

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67062265.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We show here that 2′‐deoxyadenosine (2′‐dAdo) but not adenosine was toxic to chromaffin cells of 3–4‐week‐old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3‐day period in the presence of 100 µM2′‐dAdo plus 3 µMdeoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase‐mediated nick end labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis‐like cell death by 2′‐dAdo. Lethal effects of 2′‐dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2′‐dAdo‐prompted cell death was not prevented by inhibitors of nucleoside transporter (3 µMdilazep or 1 µMnitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 µMuridine or 100 µM2′‐deoxycytidine), or 5 mMnicotinamide. Cells incubated with 2′‐dAdo (100 and 300 µM) showed a three‐ and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2′‐dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2′‐dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67062273.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Exposure of cultured cerebellar granule cells to 100 µMglutamate plus glycine in the absence of Mg2+causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca2+deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca2+concentration), and extensive cell death. Glutamate‐evoked Ca2+deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca2+transport, allows glutamate‐exposed cells to maintain a high ATP/ADP ratio while accumulating little45Ca2+and maintaining a low bulk cytoplasmic free Ca2+concentration determined by fura‐2. It is concluded that mitochondrial Ca2+accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca2+flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca2+accumulation in a microdomain accessible to the mitoch

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67062282.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The specific opioid receptor antagonist naloxone attenuates the behavioral and neurochemical effects of amphetamine. Furthermore, the amphetamine‐induced increase in locomotor activity is attenuated by intracisternally administered naltrindole, a selective δ‐opioid receptor antagonist, but not by the irreversible μ‐opioid receptor antagonist β‐funaltrexamine. Therefore, this research was designed to determine if naltrindole would attenuate the neurochemical response to amphetamine as it did the behavioral response. In vivo microdialysis was used to monitor the change in extracellular concentrations of dopamine in awake rats. Naltrindole (3.0, 10, or 30 µg) or vehicle was given 15 min before and β‐funaltrexamine (10 µg) or vehicle 24 h before the start of cumulative dosing, intracisternally in a 10‐µl volume, while the rats were lightly anesthetized with methoxyflurane. Cumulative doses of subcutaneousd‐amphetamine (0.0, 0.1, 0.4, 1.6, and 6.4 mg/kg) followed pretreatment injections at 30‐min intervals. Dialysate samples were collected every 10 min from either the striatum or nucleus accumbens and analyzed for dopamine content by HPLC. Amphetamine dose‐dependently increased dopamine content in both the striatum and nucleus accumbens, as reported previously. Naltrindole (3.0, 10, and 30 µg) significantly reduced the dopamine response to amphetamine in the striatum. In contrast, 30 µg of naltrindole did not modify the dopamine response to amphetamine in the nucleus accumbens. On the other hand, β‐funaltrexamine (10 µg) had no effect in the striatum but significantly attenuated the amphetamine‐induced increase in extracellular dopamine content in the nucleus accumbens. These data suggest that δ‐opioid receptors play a relatively larger role than μ‐opioid receptors in mediating the amphetamine‐induced increase in extracellular dopamine content in the striatum, whereas μ‐opioid receptors play a larger role in media

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67062292.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The brain is a rich source of the lipid biomediator lysophosphatidic acid, and lysophosphatidic acid levels can significantly increase following brain trauma. Responses of primary rat brain astrocytes to this novel lipid are defined in the current study. Treatment of cells with lysophosphatidic acid resulted in a time‐ and dose‐dependent inhibition of glutamate uptake. Inhibition of glutamate uptake was specific because the related phospholipids, phosphatidic acid, lysophosphatidylcholine, and lysophosphatidylglycerol, did not inhibit this uptake under comparable conditions, i.e., treatment with 10 µMlipid for 30 min. Lysophosphatidic acid treatment of cells resulted in an increase in lipid peroxidation, as measured by the thiobarbituric acid assay. This increase in content of thiobarbituric acid‐reactive substances was largely inhibited by treatment with dithiothreitol or propyl gallate; however, such treatment did not affect the lysophosphatidic acid‐induced inhibition of glutamate uptake. Lysophosphatidic acid also inhibited glucose uptake with a dose‐response curve that paralleled the inhibition of glutamate uptake. By impairing uptake of glutamate by astrocytes, lysophosphatidic acid may exacerbate excitotoxic processes in various neurodegenerative

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67062300.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The src family protein tyrosine kinases (PTKs) are nonreceptor kinases. Some PTKs of this family are ubiquitously expressed, whereas others have a more restricted expression, as in neurons. Lymphoid cell kinase (lck) p56lck is highly expressed in tissues of lymphoid origin and believed to be specific for hematopoietic cells. Reports suggesting that CD4 is expressed in neurons prompted us to analyze the possibility that p56lck is also expressed in these cells. By western blot and immunoprecipitations using anti‐lck antibody, an lck‐like protein was detected in lysates from primary cultures of rat cerebellar granular neurons. This 56‐kDa phosphoprotein was autophosphorylated in vitro and also phosphorylated enolase, similarly to p56lck. It was shown to be located actually in the neurons by immunocytofluorescence. Partial proteolysis mapping showed that the 56‐kDa phosphoprotein had a peptide pattern very similar to the p56lck protein. Retrotranscription‐PCR allowed the detection of an lck RNA in the neurons. The lck kinase domain was completely identical to the lymphocyte lck kinase domain, but the 5′ end was modified in the neurons. These results show that p56lck is not lymphoid specific as is widely believed; its expression in neurons might underlie the toxicity of the HIV glycoprotein gp120

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67062306.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY