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)

We have constructed stable cell lines expressing transporters for dopamine (DA), norepinephrine (NE), and serotonin (5-HT) by transfection with cloned cDNAs. The parental LLC-PK1 cell does not express any of these neurotransmitter transporters. Therefore, monoamine transport activities in each of these cell lines are due to the transfected DNA only, allowing comparison in the same background. Drug inhibition profiles for each cell line are distinct and as expected for each transporter. LLC-NET and LLC-DAT cells transported both NE and DA and both cell types exhibited a lower KM for DA transport than for NE transport. Analysis of Vmax data for LLC-NET cells suggests that substrate is bound to the NE transporter during the rate-limiting step(s) in transport. The cocaine analog 2-beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binds to each cell type, and is displaced by transport substrate in each case. Binding and transport measurements on parallel cell cultures allowed estimation of turnover numbers for norepinephrine, dopamine, and serotonin transporters. All three transporters require external Na+ and Cl-. The Na+ concentration dependence suggests that a single Na+ ion is involved in transport catalyzed by norepinephrine and serotonin transporters while more than one Na+ ion participate in transport mediated by the dopamine transporter.
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PMID:Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence. 812 21

The successful generation and functional expression of a series of recombinant chimeric transporters, in which distinct functional properties of NET and DAT are exchanged, have allowed the assignment of a number of important functional properties of MPP+ and antidepressant-sensitive catecholamine transporters to specific domains within their primary structure. These studies are the first comprehensive structure-function analysis of members of the rapidly growing superfamily of Na+/Cl- carriers using chimeric transporters. This represents the first step in identifying the specific structural or regulatory determinants that differentiate NET and DAT. An appreciation of the potentially distinct sites for substrate recognition, translocation, and transport inhibition of NET and DAT may facilitate the development of more selective drugs for the treatment of stimulant addiction, human depression, and other affective disorders.
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PMID:Discrete structural domains and cell-specific expression determine functional selectivity of the dopamine and norepinephrine transporters. 878 50

We have synthesized several derivative of dl-threo-methylphenidate (Ritalin) bearing substituents on the phenyl ring. IC50 values for binding these compounds to rat brain monoamine transporters were assessed using [3H]WIN 35,428 (striatal membranes, dopamine transporters, DAT), [3H]nisoxetine (frontal cortex membranes, norepinephrine transporters, NET) and [3H]paroxetine (brain stem membranes, 5HT transporters, 5HTT). Affinities (1/Ki) decreased in the order: DAT > NET >> 5HTT. Substitution at the para position of dl-threo-methylphenidate generally led to retained or increased affinity for the dopamine transporter (bromo > iodo > methoxy > hydroxy). Substitution at the meta position also increased affinity for the DAT (m-bromo > methylphenidate; m-iodo-p-hydroxy > p-hydroxy). Substitution at the ortho position with bromine considerably decreased affinity. Similar IC50 values for binding of o-bromomethylphenidate to the dopamine transporter were measured at 0, 22 and 37 degrees. N-Methylation of the piperidine ring of methylphenidate also considerably reduced affinity. The dl-erythro isomer of o-bromomethylphenidate did not bind to the DAT (IC50 > 50,000 nM). Affinities at the dopamine and norepinephrine transporters for substituted methylphenidate derivatives were well correlated (r2=0.90). Abilities of several methylphenidate derivatives to inhibit [3H]dopamine uptake in striatal synaptosomes corresponded well with inhibition of [3H]WIN 35, 428 binding. None of the compounds examined exhibited significant affinity to dopamine D1 or D2 receptors (IC50 > 500 or 5,000 nM, respectively), as assessed by inhibition of binding of [3H]SCH 23390 or [123I]epidepride, respectively, to striatal membranes.
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PMID:Affinities of methylphenidate derivatives for dopamine, norepinephrine and serotonin transporters. 878 5

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

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

We investigated the gene expression of three monoamine transporters (norepinephrine transporter, NET; serotonin transporter, SERT; and dopamine transporter, DAT) in the rat superior cervical ganglion (SCG). Most of principal ganglion neurons abundantly expressed NET mRNA. In addition, about 30% of principal ganglion neurons also expressed SERT mRNA. However, DAT mRNA expression was not observed there. These results suggest that serotonin as well as norepinephrine works as a neurotransmitter in a subset of principal ganglion neurons.
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PMID:Expression of norepinephrine and serotonin transporter mRNAs in the rat superior cervical ganglion. 1010 Dec 35

Two serine residues in the beta-adrenergic receptor (beta-AR) have been proposed to form hydrogen bonds with the catechol moiety of the ligand and contribute to the activation of the receptor. These conserved serine residues in the dopamine (DA) and norepinephrine transporters (DAT and NET, respectively) have also been shown to affect substrate transport in the rat DAT. In the present work, hydrogen bonding interactions between the corresponding serine residues in the human NET (hNET), 354 and 357, and the hydroxyl groups on the substrate were systematically evaluated by examining the transport and binding properties of DA and several single hydroxyl analogues of DA at wild-type and serine-to-alanine-substituted transporters. A comparison of [3H]nisoxetine binding at the serine 354 mutant, in which K(D) increased 70-fold from the wild-type value, with the binding of DA, m-tyramine (m-TYR), and p-tyramine (p-TYR) at mutant 354, where the increase in Ki was less dramatic, revealed that serine 354 is more influential in inhibitor than substrate binding. The binding of m-TYR and p-TYR at the serine 354 and serine 357 mutants did not show a direct interaction between one serine and one substrate catechol hydroxyl group. DA, m-TYR, and p-TYR binding affinity did not deviate from the wild-type value at the serine 357 and double mutant transporters. At these two transporters, however, the Km of DA uptake increased, suggesting that the roles of serine 357 and serine 354 in substrate transport are different from their roles in binding. The K'm for induced efflux of DA decreased at the serine 357 mutant compared with the wild-type, whereas the K'm at the serine 354 mutant was the same as that of the wild-type. Further investigation of the role of substrate hydroxyls in the transport process revealed no difference between the transport of m-TYR or p-TYR, as measured indirectly through their induced efflux of DA, at any of the mutants. Although these serines are influential in inhibitor and substrate binding to the transporter and substrate uptake and efflux, they do not appear to be involved in a direct hydrogen bond interaction with substrate, suggesting that the pattern of distinct hydrogen bonding interactions at the beta-AR does not exist at the hNET.
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PMID:Effects of serine mutations in transmembrane domain 7 of the human norepinephrine transporter on substrate binding and transport. 1042 62

A new radioligand, 5-iodo-2-[[2-2-[(dimethylamino)methyl]phenyl]thio]benzyl alcohol ([(123)I]IDAM), has been developed for selective single-photon emission tomography (SPET) imaging of SERT. In vitro binding studies suggest a high selectivity of IDAM for SERT (K(i)=0.097 nM), with considerably lower affinities for norepinephrine and dopamine transporters (NET K(i)= 234 nM and DAT K(i)>10 microM, respectively). In this study the biodistribution of SERT in the baboon brain was investigated in vivo using [(123)I]IDAM and SPET imaging. Dynamic sequences of SPET scans were performed on three female baboons (Papio anubis) after injection of 555 MBq of [(123)I]IDAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 90-120 min after injection of [(123)I]IDAM. Similar studies were performed using a NET inhibitor, nisoxetine, and a DAT blocker, methylphenidate. After 60-120 min, the regional distribution of tracer within the brain reflected the characteristic distribution of SERT, with the highest uptake in the midbrain area (hypothalamus, raphe nucleus, substantia nigra), and the lowest uptake in the cerebellum (an area presumed free of SERT). Peak specific binding in the midbrain occurred at 120 min, with a ratio to the cerebellum of 1.80+/-0.13. At 30 min, 85% of the radioactivity in the blood was metabolite. Following injection of a competing SERT ligand, (+)McN5652, the tracer exhibited rapid washout from areas with high concentrations of SERT (dissociation rate constant in the midbrain, averaged over three baboons, k(off)=0. 025+/-0.002 min(-1)), while the cerebellar activity distribution was undisturbed (washout rate 0.0059+/- 0.0003 min(-1)). Calculation of tracer washout rate pixel-by-pixel enabled the generation of parametric images of the dissociation rate constant. Similar studies using nisoxetine and methylphenidate had no effect on the distribution of [(123)I]IDAM in the brain. These results suggest that [(123)I]IDAM is suitable for selective SPET imaging of SERT in the primate brain, with high contrast, favorable kinetics, and negligible binding to either NET or DAT.
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PMID:Single-photon emission tomography imaging of serotonin transporters in the non-human primate brain with the selective radioligand [(123)I]IDAM. 1043 98

3-Phenyltropane analogues of cocaine are useful neurobiologic tools for examining mechanisms of neurotransmitter transporters and psychostimulant drugs. They are also potential substitute medications for psychostimulant abuse. In this study, 18 3-phenyltropane analogues were characterized in uptake and binding studies at dopamine (DAT), norepinephrine (NET) and serotonin (SERT) transporters from the rat, and in binding at DAT in rat, rhesus monkey, and human brain tissue. In rat brain tissue, potency in inhibiting uptake generally correlated with the potency in inhibiting binding at all three transporters suggesting that none of these compounds have antagonist properties. At the DAT, there was a significant correlation of inhibitory potencies between the rat and monkey, the monkey and human, and the rat and human transporters although some compounds showed some species difference. These findings suggest that with regard to the 3-phenyltropane series, there is generally little pharmacologic difference between DATs from the three species examined, although binding data from rat may not be a perfect predictor of uptake inhibition in human.
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PMID:Studies of selected phenyltropanes at monoamine transporters. 1046 87

The mechanism of release mediated by the human dopamine and norepinephrine transporter (DAT and NET, respectively) was studied by a superfusion technique in human embryonic kidney 293 cells stably transfected with the respective transporter cDNA and loaded with the metabolically inert substrate [(3)H]1-methyl-4-phenylpyridinium. Release was induced by amphetamine, dopamine, and norepinephrine or by lowering the sodium or chloride concentration in the superfusion buffer (iso-osmotic replacement by lithium and isethionate, respectively). Efflux of [(3)H]1-methyl-4-phenylpyridinium was analyzed at 30-s time resolution. In both transporters, release induced by the substrates amphetamine, dopamine, and norepinephrine followed the same time course as release induced by the removal of chloride and was faster than that caused by the removal of sodium. In the presence of low sodium (DAT: 10 mM; NET: 5 mM) none of the substrates was able to induce release from either type of cell, but adding back sodium to control conditions promptly restored the releasing action. In the presence of low chloride (DAT: 3 mM; NET: 2 mM), however, amphetamine as well as the catecholamines stimulated release from both types of cell. In contrast with the ion dependence of release observed in superfusion experiments, uptake initial rates of substrates at concentrations used in release experiments were the same or even higher at low sodium than at low chloride. The results indicate a decisive role of extracellular sodium for carrier-mediated release unrelated to the sodium-dependent uptake of the releasing substrate, and suggest a release mechanism different from simple exchange diffusion considering only the amines as substrates.
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PMID:Ion dependence of carrier-mediated release in dopamine or norepinephrine transporter-transfected cells questions the hypothesis of facilitated exchange diffusion. 1053 12


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