摘要:

Abstract:PCR was used to isolate identical partial cDNA clones encoding a serotonin 5‐HT3receptor subunit from rat nodose and superior cervical ganglia. The amino acid sequence predicted from these clones, extending from the putative transmembrane domain I to the stop codon, demonstrated a 93% homology with the 5‐HT3receptor A (R‐A) subunit cloned from NCB 20 hybridoma mouse neuroblastoma/Chinese hamster embryonic brain cells. Comparison of the sequences of the rat gene and cDNA encoding this subunit revealed a five amino acid deletion, GSLLP, located within the putative second intracellular loop of the receptor subunit. This deletion was shown to occur at an intron/exon junction. Therefore, alternative splicing was probably responsible for the presence of short (5‐HT3R‐AS) and long (5‐HT3R‐AL) forms of 5‐HT3R‐A mRNA in these ganglia. PCR experiments, with specific primers located upstream and downstream of the GSLLP deletion, were used to detect reverse transcribed 5‐HT3R‐A mRNAs. A short fragment (92 bp), corresponding to the deleted form, and a long fragment (107 bp), corresponding to the nondeleted form, were amplified from various regions of the CNS and peripheral ganglia of the rat, as well as from NG108‐15 hybridoma cells. In the adult rat, the ratio of the two forms varied very little from one tissue to another, the long form corresponding to only ∼10% of the total 5‐HT3R‐A mRNA. Study of their respective distributions during ontogeny demonstrated a differential expression of the short and long forms in some tissues during late embryonic development, at embryonic day 17 (E17) or E20. In particular, the long form amounted to about one‐third of the total 5‐HT3R‐A mRNA in the cerebral cortex and hippocampus at E17, and this proportion reached 50 and 75% in the superior cervical ganglion and nodose ganglion, respectively, at E20. These data indicate that alternative splicing of the 5‐HT3R‐A mRNA is regulated in the CN

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65020475.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Both, 2,500‐ and 6,000‐nucleotide (nt) mRNAs are generated by alternative splicing of the primary transcript from the human gene for choline acetyltransferase (ChAT), the 68‐kDa enzyme that synthesizes acetylcholine. In vitro translation of cRNA derived from a clone of the 2,500‐nt mRNA produced a protein with ChAT activity demonstrating that this transcript encodes the human ChAT enzyme. An antibody directed against a unique amino acid sequence predicted from the 6,000‐nt ChAT gene transcript identified a 27‐kDa protein on immunoblots of human nucleus basalis proteins. This protein was further shown to cross‐react with antibodies prepared against the 68‐kDa human ChAT enzyme. Gel‐filtration chromatography of human nucleus basalis proteins demonstrated that the 27‐kDa protein does not have ChAT activity, which eluted as a single peak coincident with that of the 68‐kDa enzyme. The 27‐kDa protein was, however, shown to colocalize with the ChAT enzyme in cholinergic neurons of the human spinal cord using immunoh

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65020484.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Agonist‐induced regulation of adrenergic receptors (ARs) has an important role in controlling physiological functions in response to changes in catecholamine stimulation. We previously generated transgenic mice expressing phenylethanolamineN‐methyltransferase (PNMT) under the control of a human dopamine β‐hydroxylase gene promoter to switch catecholamine specificity from the norepinephrine phenotype to the epinephrine phenotype. In the present study, we first examined changes in catecholamine metabolism in peripheral tissues innervated by sympathetic neurons of the transgenic mice. In the transgenic target tissues, a high‐level expression of PNMT led to a dramatic increase in the epinephrine levels, whereas the norepinephrine levels were decreased to 48.6–87.9% of the nontransgenic control levels. Analysis of plasma catecholamines in adrenalectomized mice showed large amounts of epinephrine derived from sympathetic neurons in the transgenic mice. Subsequently, we performed radioligand binding assays with (−)‐[125I]iodocyanopindolol to determine changes in binding sites of β‐AR subtypes. In transgenic mice, the number of β2‐AR binding sites was 56.4–74.9% of their nontransgenic values in the lung, spleen, submaxillary gland, and kidney, whereas the β1‐AR binding sites were regulated in a different fashion among these tissues. Moreover, northern blot analysis of total RNA from the lung tissues showed that down‐regulation of β2 binding sites was accompanied by a significant decrease in steady‐state levels of the receptor mRNA. These results strongly suggest that alteration of catecholamine specificity in the transgenic sympathetic neurons leads to regulated expression of the β‐AR

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65020492.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:To investigate the regulation of norepinephrine transporter mRNA in vivo, we analyzed the effects of reserpine on its expression in the rat adrenal medulla and locus ceruleus. First, PCR was used to clone a 0.5‐kb rat cDNA fragment that exhibits 87% nucleotide identity to the corresponding human norepinephrine transporter cDNA sequence. In situ, the cDNA hybridizes specifically within norepinephrine‐secreting cells, but in neither dopamine nor serotonin neurons, suggesting strongly it is a partial rat norepinephrine transporter cDNA. Reserpine, 10 mg/kg administered 24 h premortem, decreased steady‐state levels of norepinephrine transporter mRNA in the adrenal medulla by ∼65% and in the locus ceruleus by ∼25%, as determined by quantitative in situ hybridization. Northern analysis confirmed the results of the in situ hybridization analysis in the adrenal medulla but did not detect the smaller changes observed in the locus ceruleus. Both analyses showed that reserpine increased tyrosine hydroxylase expression in the adrenal medulla and locus ceruleus. These results suggest that noradrenergic neurons and adrenal chromaffin cells can coordinate opposing changes in systems mediating catecholamine uptake and synthesis, to compensate for catecholamine

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65020502.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Expression of the gene encoding the neurotransmitter biosynthetic enzyme dopamine β‐hydroxylase (DBH) is regulated in a tissue‐specific pattern, and transcription is influenced by environmental stimuli. Using the promoter proximal region of the rat DBH gene and nuclear extracts from SHSY‐5Y neuroblastoma cells, a DNA‐protein complex was identified that is competitive with oligonucleotides containing the recognition site of transcription factor AP‐2. DNase footprint analysis identified an AP‐2 binding site between −136 and −115 of the DBH promoter. Mutation of that AP‐2 site results in a sevenfold reduction of basal reporter gene expression, but second messenger‐stimulated activity is retained. Cotransfection of an AP‐2 expression vector and a DBH promoter‐reporter construct into cultured cells results in a sixfold stimulation of reporter gene expression, demonstrating the ability of AP‐2 totrans‐activate the DBH promoter. These results identify a new regulatory element on the rat DBH gene and suggest that the AP‐2 site plays a role in maintaining bas

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65020510.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH‐SY‐5Y human neuroblastoma cells. SH‐SY‐5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum‐containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell‐permeant cysteine protease inhibitorsN‐acetyl‐leucyl‐leucyl‐norleucinal (“C1”) and calpeptin or by the phospholipid‐mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti‐calpain antisera. Calpain inhibition in intact cells was confirmed by immunoblot analysis showing inhibition of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule‐associated protein 1. The above inhibitory peptides and antiserum did not induce neurites in medium containing serum but lacking hirudin, suggesting that increased surface protein adhesiveness is a prerequisite for enhancement of neurite outgrowth by calpain inhibition. Treatment of cells with the PKC inhibitor H7, staurosporine, or sphingosine induced neurite outgrowth independently of serum concentration. Because calpain is thought to regulate PKC activity, we examined this potential interrelationship during neurite outgrowth. Simultaneous treatment with calpain and PKC inhibitors did not produce additive or synergistic effects on neurite outgrowth. PKC activation by 2‐O‐tetradecanoylphorbol 13‐acetate (TPA) prevented and reversed both neurite initiation by serum deprivation and its enhancement by calpain inhibitors. Treatment of cells with the calpain inhibitor C1 retarded PKC down‐regulation following TPA treatment. Cell‐free analyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of millimolar calcium‐requiring calpain to cleave the SH‐SY‐5Y PKC regulatory subunit from the catalytic subunit, yielding a free catalytic subunit (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65020517.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:In examining steroid synthesis in the CNS, expression of the mRNAs encoding for cytochrome P450 side‐chain cleavage enzyme (P450SCC) and 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4isomerase (3β‐HSD) has been studied in the rat brain. P450SCCtransforms cholesterol into pregnenolone and 3β‐HSD transforms pregnenolone into progesterone. PCR was used to amplify cDNA sequences from total RNA extracts. Classical steroidogenic tissues, like adrenal and testis, as well as the non‐steroidogenic tissue lung have been used as controls. The expression of P450SCCand 3β‐HSD have been demonstrated by PCR in cortex, cerebellum, and spinal cord. In addition, primary cultures of rat cerebellar glial cells and rat cerebellar granule cells were found to express P450SCCand 3β‐HSD at comparable levels. Furthermore, three of the four known isoenzymes of 3β‐HSD were identified, as determined using selective PCR primers coupled with discriminative restriction enzymes and sequencing analysis of the amplified brain products. Using RNA probes, in situ hybridization indicated that P450SCCand 3β‐HSD are expressed throughout the brain at a low level and mainly in white matter. Enrichment of glial cell cultures in oligodendrocytes, however, does not increase the relative abundance of P450SCCand 3β‐HSD mRNA detected by PCR. This discrepancy suggests that the developmental state of cultured cells and their intercellular environment may be critical for regulating the expression of these enzymes. These findings support the proposal that the brain apparently has the capacity to synthesize progesterone from cholesterol, through pregnenolone, but that the expression of these enzymes appears to be quite low. Furthermore, the identification of these messages in cerebellar granule cell cultures implies that certain neurons, in addition to glial cells, may expr

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65020528.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The human neuroblastoma cell line SKNBE can be differentiated either by serum removal or by adding to the culture medium different morphogens, for instance, retinoic acid (RA), cyclic AMP derivatives, and phorbol esters. Both the differentiated and undifferentiated cells express the two types of membrane tumor necrosis factor (TNF) receptors (TNFRs) of 55 and 75 kDa (p55 and p75 TNFR, respectively) and also their soluble forms. After RA addition the number of the surface TNFRs per cell is increased approximately twofold, but the kinetics of expression are different, depending on the receptor type. The level of the mRNAs of 2.4 and 4.2 kb, which, respectively, encode the p55 and p75 TNFRs, is also increased during the time course of differentiation, and the kinetics of their expression are biphasic. In contrast, the number of TNFRs and the level of their encoding mRNAs remain unchanged after exposure of the cells to both a phorbol and a cyclic AMP derivative.

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65020537.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Murine neuroblastoma × embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human β2‐adrenoceptor under the control of a β‐actin promoter. Two clones were selected for detailed analysis: D1, which expressed some 12.7 pmol/mg of membrane protein, and L9, which expressed 1.2 pmol/mg of membrane protein of the receptor. Incubation with the β‐adrenoceptor agonist isoprenaline resulted in stimulation of adenylyl cyclase activity in both of the clones, whereas no such activation was observed in wild‐type NCB20 cells. The EC50for isoprenaline stimulation of adenylyl cyclase activity in membranes of clone D1 (0.8 nM) was significantly lower, however, than in membranes of clone L9 (10.4 nM). Although the maximal adenylyl cyclase stimulation by isoprenaline was similar in both clones, D1 had a higher basal activity. Immunoblotting studies with specific antipeptide antisera directed against various G protein α subunits showed that treatment of the cells with isoprenaline resulted in a 35% reduction in the membrane‐associated levels of Gsα in membranes of clone L9 cells and a 50% reduction in Gsα levels in membranes prepared from clone D1. Isoprenaline treatment had no effect on the levels of Gsα in wild‐type NCB20 cells, and such treatment had no effect on the levels of other G protein α subunits such as Gq/G11and Gi2 in any of the cell lines investigated. Time course analysis revealed that half‐maximal loss of Gsα in clone D1 was achieved within 1–2 h of addition of agonist. Dose‐response curves to isoprenaline in clone D1 indicated that half‐maximal down‐regulation of Gsα was produced by ∼1 nMagonist. Measurement of GsmRNA levels in both clones, however, using both reverse transcriptase‐polymerase chain reaction and northern blotting revealed no significant change f

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65020545.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The mixed function oxidase system consists of NADPH‐cytochrome P450 reductase (P450 reductase) and various isoforms of cytochrome P450 (P450), which can catalyze the oxidation of a broad range of endogenous and exogenous compounds. In this study, we examined the rat glioma C6 cell line for the presence of P450 reductase and three isozymes of cytochrome P450, 1A1, 2B1, and 2B2, by reverse transcription followed by PCR (RT‐PCR). Rat glioma C6 cells were treated with hepatic P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). Cytochromes P450 1A1, P450 2B1, and P450 2B2, and P450 reductase, were detected in all the different treatment groups. Restriction digestion was used to confirm the PCR fragments and the expected digestion products were obtained. The induction of P450 1A1 and 2B was quantified using competitive PCR. Ten‐ and five‐fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected by competitive PCR. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 spectra with absorption at 450 nm. EthoxyresorufinO‐deethylase activity (11.5 ± 1.7 pmol/min/mg of microsomal protein) and pentoxyresorufinO‐dealkylation activity (8.9 ± 1.4 pmol/min/mg of microsomal protein) confirmed the induction of P450 1A and 2B at the protein level in response to BA or PB treatments, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active mixed function oxidase system that can be induced by hepatic

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.65020554.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY