Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antibodies have been raised against synthetic peptides derived from the predicted primary sequence of the human cocaine- and antidepressant-sensitive norepinephrine (NE) transporter (NET). One antibody (N430), raised and purified against a putative intracellular human norepinephrine transporter (hNET) epitope, detects hNET expression in a stably transfected cell line (LLC-NET) by indirect immunofluorescence only in the presence of detergent, while no immunoreactivity is observed in either the parental cells (LLC-PK1) or in LLC-NET cells incubated with preimmune sera or peptide absorbed antibody. N430 immunoblots of LLC-NET cell extracts reveal two major immunoreactive hNET species in these cells, migrating at 80 and 54 kDa, respectively. Pulse-chase N430 immunoprecipitation studies confirm that the 54-kDa species is a transient, glycosylated intermediate of a longer lived, more highly glycosylated protein with an apparent M(r) of 80,000. In contrast, a 54-kDa species is the primary hNET product in vaccinia virus T7-infected HeLa cells, transiently transfected with hNET cDNA. PNGase F digestion of extracts prepared from LLC-NET- and hNET-transfected HeLa cells convert all immunoreactive species to a 46-kDa form, equivalent to that observed following incubation of whole cells with the glycosylation inhibitor tunicamycin. As transiently transfected HeLa and stable LLC-NET cells exhibit a pharmacologically similar NE transport activity, it appears likely that the additional glycosylation evident in the stable line does not contribute significantly to antagonist sensitivity. On the other hand, NE transport and antagonist ([125I]RTI-55) binding assays on whole LLC-NET cells treated with tunicamycin reveal a pronounced reduction in NE transport activity and hNET membrane density paralleled by an inability of NET proteins to replenish the higher M(r) hNET pool. These findings suggest an obligate role for N-linked glycosylation in hNET biosynthetic maturation, stability, and functional expression. In summary, N430 antibody is a useful tool for the visualization and characterization of hNET gene products and has permitted the first direct evaluation of biosynthetic steps leading to functional catecholamine transporter expression.
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PMID:Human norepinephrine transporter. Biosynthetic studies using a site-directed polyclonal antibody. 816 33

Metaiodobenzylguanidine (MIBG) is taken up by sympathetic neurons, but the precise mechanism of uptake has not been elucidated. Uptake of monoamines by presynaptic neurons is mediated by plasma membrane proteins, the monoamine transporters. The human norepinephrine transporter (hNET), the bovine dopamine transporter (bDAT) and the rat serotonin transporter (r5HTT) have been cloned, sequenced and expressed in various cell lines. This study involves the measurement of MIBG uptake by cell lines that have been transfected with complementary DNAs encoding these monoamine transporters. At 20 nM MIBG, hNET transfected cells demonstrate a ninefold greater uptake of MIBG than nontransfected cells. MIBG uptake in hNET transfected cells is inhibited by 3 x 10(-6) M norepinephrine (87% inhibition) and by hNET transport inhibitors: 10(-7) M desipramine (94% inhibition) and 10(-7) M mazindol (97% inhibition). hNET transfected cells exhibit a Km for MIBG transport of 264 nM. Percent nonspecific uptake rises with increasing concentrations of MIBG while specific uptake is saturable. There is no significant uptake by bDAT or r5HTT. The NET appears to be responsible for the specific uptake of MIBG.
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PMID:Evaluation of metaiodobenzylguanidine uptake by the norepinephrine, dopamine and serotonin transporters. 831 92

The role of N-glycosylation in the expression, stability, and ligand recognition by the cocaine- and antidepressant-sensitive human norepinephrine transporter (hNET) was assessed in stably and transiently transfected cell lines. The use of hNET-specific antibodies and the membrane-impermeant biotinylating reagent sulfosuccinimidobiotin establishes that treatment of stably transfected LLC-PK1 cells with tunicamycin depletes surface membranes of mature hNET glycoproteins, which is consistent with a failure of less stable, nonglycosylated subunits to replenish surface compartments. To determine whether N-glycosylation plays a direct role in hNET stability, surface expression, and ligand recognition, we mutated the three hNET canonical N-glycosylation sites (hNETN184, 192, 198Q) and transiently expressed the mutant cDNA in parallel with the parental hNET construct in HeLa and COS cells. hNETN184, 192, 198Q protein exhibited increased electrophoretic mobility (approximately 46 kDa), similar to that of enzymatically N-deglycosylated hNET protein, which confirms the use of canonical sites in the second extracellular loop of the transporter. hNETN184, 192, 198Q protein in HeLa and COS extracts was reduced approximately 50% relative to hNET protein in parallel transfections, demonstrated to arise from a reduction in transporter half-life, which is consistent with the proposed role of N-glycosylation in hNET stability. Both HeLa and COS cells transfected with hNETN184, 192, 198Q exhibit a significantly greater reduction in transport activity than can be accounted for by losses in either total or surface NET protein. Furthermore, sensitivity of catecholamine transport to unlabeled substrate and antagonists was unchanged in the mutant, suggesting that residual nonglycosylated surface hNETs execute a key step in the transport cycle after ligand recognition with reduced efficiency.
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PMID:Inability to N-glycosylate the human norepinephrine transporter reduces protein stability, surface trafficking, and transport activity but not ligand recognition. 870 Jan 33

Rotating disk electrode (RDE) voltammetry is applied to the measurement of the transport of the catecholamine neurotransmitters norepinephrine (4-(2-amino-1-hydroxyethyl)-1,2-benzenediol, NE) and dopamine (3,4-dihydroxyphenethylamine, DA) in suspensions of LLC-NET cells, a line of porcine kidney cells expressing the human norepinephrine transporter (hNET). Initial rate of transport was assessed by following the initial decrease in neurotransmitter after its addition to the cell suspension, as measured by the decrease in oxidation current at +0.45 V vs Ag/AgCl. The initial rate of norepinephrine uptake was saturable, with Vmax and KM of 197 +/- 17 amol min-1 cell-1 and 1.64 +/- 0.46 microM, respectively. The RDE method also allows observation of outward transport (efflux) of the DA or NE previously taken up by the cells. Outward transport was induced by the addition of either d-amphetamine (d-AMPH) or p-tyramine (4-hydroxyphenethylamine, p-TYR), which are also substrates for the NE transporter. The technique was also used to monitor accelerated NE uptake by cells preloaded with p-TYR, a phenomenon distinguishing carriers from channels. Together, these findings document the utility of RDE for the nonisotopic measurement of neurotransmitter influx and efflux from transfected mammalian cells.
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PMID:Human norepinephrine transporter kinetics using rotating disk electrode voltammetry. 879 28

We have previously reported that chronic elevation of insulin in the CNS of rats results in opposing changes of the mRNA expression for the norepinephrine transporter (NET; decreased) and the dopamine transporter (DAT; increased). In the present study we tested the hypothesis that a chronic depletion of insulin would result in opposite changes of NET and DAT mRNA expression, from those observed with chronic elevation of insulin. Rats were treated with streptozotocin to produce hypoinsulinemic diabetes. One week later, steady state levels of mRNA were measured by in situ hybridization for NET in the locus coeruleus (LC) and for DAT in the ventral tegmental area/substantia nigra compacta (VTA/SNc). The mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme for NE and DA synthesis, was measured in these same brain regions. In the diabetic animals, NET mRNA was significantly elevated (159 +/- 22% of average control level) while DAT mRNA was non-significantly decreased (78 +/- 9% of average control level). Additionally, TH mRNA was significantly altered in both the LC (131 +/- 11% of average control level) and VTA/SNc (79 +/- 5% of average control level). We conclude that endogenous insulin is one physiological regulator of the synthesis and re-uptake of NE and DA in the CNS.
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PMID:Diabetes causes differential changes in CNS noradrenergic and dopaminergic neurons in the rat: a molecular study. 893 Mar 8

The purpose of this study was to determine the primary form of human placental norepinephrine transporter (hNET) mRNA expressed in the human placenta and to compare the level of expression in normal pregnancies and in pregnancies complicated by drug exposure or other forms of physiological derangement. We used the hNET cDNA to measure RNA extracted from placenta and examined placental RNA following complicated and uncomplicated pregnancies. To compare transporter expression and its relation to fetal condition at birth, umbilical arterial plasma catecholamine levels, umbilical arterial blood gases and placental transporter mRNA level were compared by linear regression analysis. Uncomplicated pregnancies had a higher level of placental norepinephrine transporter mRNA than complicated pregnancies. An inverse relationship between umbilical cord norepinephrine level and transporter expression was demonstrated. We conclude that placental transporter expression represents an important and newly described metabolic function of the placenta. Placental catecholamine clearance mediated via the placental NET may be important in the pathophysiology of disorders associated with placental dysfunction, impaired placental blood flow or intrauterine growth retardation. This may also explain the adverse effects of drugs, such as cocaine, which block catecholamine transport.
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PMID:Human placental norepinephrine transporter mRNA: expression and correlation with fetal condition at birth. 908 83

Quantitative in situ hybridization was utilized to map the distribution and abundance of the serotonin, dopamine and norepinephrine transporter (SERT, DAT and NET, respectively) mRNAs. SERT mRNA was quantified within the dorsal raphe (DR) and the median raphe (MR), DAT mRNA within the ventral tegmental area -substantia nigra (VTA-SN) region and NET mRNA within the locus coeruleus (LC). SERT mRNA expression within the raphe complex was organized into distinct subregional domains with the rank order of mRNA abundance: ventromedial (vm) DR > dorsomedial (dm) DR > MR > dorsolateral (dl) DR. The relative abundance of DAT mRNA also varied across subregions: SN pars compacta > the parabrachial pigmentosis (PBP) > the intrafascicular (IF). The effects of a 'binge' paradigm of cocaine administration on SERT, DAT and NET mRNA abundance were compared in the brains of behaviorally sensitized rats. Cocaine significantly decreased the abundance of the SERT mRNA within the dlDR and DAT mRNA abundance within the SNc and the PBP, and increased the abundance of the NET mRNA within the LC. Finally, correlational analysis indicated that post-cocaine levels of DAT, SERT and NET mRNAs were not associated with cocaine-induced sensitization.
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PMID:Serotonin, dopamine and norepinephrine transporter mRNAs: heterogeneity of distribution and response to 'binge' cocaine administration. 938 68

The norepinephrine (NE) transporter (NET), a target of many clinically prescribed antidepressants, regulates noradrenergic neurotransmission by efficiently clearing NE from synaptic spaces after release. To advance our understanding of NET gene structure, regulation, and potential associations with complex behavioral trait loci, we amplified a mouse norepinephrine transporter (mNET) cDNA from placenta total RNA and utilized mNET probes to isolate and characterize the mNET gene. Inferred translation of the major open reading frame of the mNET cDNA predicts a 617-amino acid protein with 12 putative membrane-spanning regions and 94% identity to human NET. The coding exons of the mNET cDNA were found to be spread across >36 kb of 129/Svj genomic DNA, with exon-intron boundaries bearing consensus gt/ag splice sites. Sequence upstream (202 bp) of the inferred translation initiation site matched the sequence of 5' rapid amplification of cDNA ends products from brain mRNA with no evidence for intervening introns and is preceded by a TATA box and canonical transcriptional regulatory elements that may play a role in mNET expression in vivo. Probes derived from mNET cDNA identified species-specific MspI restriction fragment length variations within the mNET gene that were utilized to position the gene (Slc6a5) to murine chromosome 8, one recombinant distal to D8Mit15. This site is within a recently defined quantitative trait locus defined for ethanol sensitivity in LSXSS recombinant inbred mice, Lore4. The status of Slc6a5 as a candidate gene for alcohol sensitivity is discussed with respect to studies noting ethanol-induced alterations in brain NE receptors, NE receptor-linked adenylate cyclase, and NE transport.
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PMID:Cloning and chromosomal mapping of the murine norepinephrine transporter. 960 88

The effects of substrates m-tyramine and beta-phenethylamine, as well as cocaine, on the DA efflux from a cell line stably expressing the human norepinephrine transporter (hNET) were investigated by using rotating disk electrode voltammetry. Both the substrates and cocaine induced apparent DA efflux in a concentration-dependent manner. Their EC50 values for inducing DA efflux were similar to their IC50 values for inhibiting DA uptake. The substrate-induced DA efflux was inhibited by various NET blockers, enhanced by raising the internal [Na+] with Na+,K+-ATPase inhibition, but was insensitive to membrane potential-altering agents valinomycin, veratridine, and high [K+]. The initial rate of m-tyramine-induced DA efflux was related to preloaded [DA] in a manner defined by a Michaelis-Menten expression. In contrast, DA efflux in the presence of cocaine displayed a much slower efflux rate, lower efficacy, was not stimulated by elevated internal [Na+], and was nonsaturable with preloaded [DA]. Single exponential kinetic analysis of the entire time course of the DA efflux showed that the apparent first-order rate constant for m-tyramine-induced DA efflux declined with increased preloaded [DA], whereas that for the DA efflux in the presence of cocaine was unchanged with varying preloaded [DA]. These results suggest that the substrates stimulate the NET-dependent DA efflux by increasing the accessibility of the NET to internal DA, whereas cocaine "uncovers" NET-independent DA efflux by reducing the accessibility of diffused/leaked external DA to the NET.
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PMID:Voltammetric studies on mechanisms of dopamine efflux in the presence of substrates and cocaine from cells expressing human norepinephrine transporter. 968 56

A small subset of neurons in the nematode Caenorhabditis elegans utilizes the catecholamine dopamine (DA) as a neurotransmitter to control or modulate movement and egg-laying. Disruption of DA-mediated behaviors represents a potentially powerful strategy to identify genes that are likely to participate in dopaminergic systems in man. In vertebrates, extracellular DA is inactivated by presynaptic DA transport proteins (DATs) that are also major targets of addictive agents, including amphetamines and cocaine. We used oligonucleotides derived from the C. elegans genomic locus T23G5.5 to isolate and characterize T23G5.5 cDNAs. Our studies predict that mRNAs from this locus encode a 615-amino-acid polypeptide with twelve stretches of hydrophobicity suitable for transmembrane domains, similar to that found in vertebrate catecholamine transporters. The inferred translation product bears highest identity (43-47%) to catecholamine (DA, norepinephrine, epinephrine) transporters within the GAT1/NET gene family and possesses conserved residues implicated in amine substrate recognition. Consistent with these findings, HeLa cells transfected with the C. elegans cDNA exhibit saturable and high affinity DA transport (Km = 1.2 microM) that is dependent on extracellular Na+ and Cl- and blocked by inhibitors of mammalian catecholamine transporters, including norepinephrine transporter- and DAT-selective antagonists, tricyclic antidepressants, and the nonselective amine transporter antagonists cocaine and D-amphetamine. These studies validate the T23G5.5 locus as encoding a functional catecholamine transporter, providing important comparative sequence information for catecholamine transporter structure/function studies and a path to identify regulators of dopaminergic signaling via genetic or pharmacologic manipulation of C. elegans cDNA in vivo.
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PMID:The Caenorhabditis elegans gene T23G5.5 encodes an antidepressant- and cocaine-sensitive dopamine transporter. 976 1


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