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Proceedings of the National Academy of... May 1994We report here on the isolation and characterization of a serotonin (5HT) transporter from Drosophila melanogaster. A 3.1-kb complementary DNA clone (dSERT) was found to...
We report here on the isolation and characterization of a serotonin (5HT) transporter from Drosophila melanogaster. A 3.1-kb complementary DNA clone (dSERT) was found to encode a protein of 622 amino acid residues with a predicted molecular mass of approximately 69 kDa and a putative transmembrane topology characteristic of cloned members of the mammalian Na+/Cl- neurotransmitter cotransporter gene family. dSERT displays highest overall amino acid sequence identity with the mammalian 5HT (51%), norepinephrine (47%), and dopamine (47%) transporters and shares with all transporters 104 absolutely conserved amino acid residues. Upon transient expression in HeLa cells, dSERT exhibited saturable, high-affinity, and sodium-dependent [3H]5HT uptake with estimated Km and Vmax values of approximately 500 nM and 5.2 x 10(-18) mol per cell per min, respectively. In marked contrast to the human SERT (hSERT), 5HT-mediated transport by dSERT was not absolutely dependent on extracellular Cl-, while the sodium-dependent uptake of 5HT was facilitated by increased extracellular Cl- concentrations. dSERT displays a pharmacological profile and rank order of potency consistent with, but not identical to, mammalian 5HT transporters. Comparison of the affinities of various compounds for the inhibition of 5HT transport by both dSERT and hSERT revealed that antidepressants were 3- to 300-fold less potent on dSERT than on hSERT, while mazindol displayed approximately 30-fold greater potency for dSERT. Both cocaine and RTI-55 inhibited 5HT uptake by dSERT with estimated inhibition constants of approximately 500 nM, while high concentrations (> 10 microM) of dopamine, norepinephrine, octopamine, tyramine, and histamine failed to inhibit transport. In situ hybridization reveals the selective expression of dSERT mRNA to specific cell bodies in the ventral ganglion of the embryonic and larval Drosophila nervous system with a distribution pattern virtually identical to that of 5HT-containing neurons. The dSERT gene was mapped to position 60C on chromosome 2. The availability of the gene encoding the unique ion dependence and pharmacological characteristics of dSERT may allow for identification of those amino acid residues and structural motifs that confer the pharmacologic specificity and genetic regulation of the 5HT transport process.
Topics: Amino Acid Sequence; Animals; Carrier Proteins; Chlorides; Cloning, Molecular; Cocaine; Drosophila Proteins; Drosophila melanogaster; Humans; In Situ Hybridization; Membrane Glycoproteins; Membrane Transport Proteins; Molecular Sequence Data; Nerve Tissue Proteins; Sequence Homology, Amino Acid; Serotonin; Serotonin Plasma Membrane Transport Proteins
PubMed: 8197200
DOI: 10.1073/pnas.91.11.5158 -
PloS One 2012The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing...
The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish). This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf), knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1), and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant) revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers.
Topics: Animal Feed; Animals; Appetite Depressants; Appetite Regulation; Cyclobutanes; Drug Evaluation, Preclinical; Feeding Behavior; Fluorescent Dyes; Fluoxetine; Gene Expression Regulation; Gene Knockdown Techniques; High-Throughput Screening Assays; Humans; Larva; Paramecium; Zebrafish
PubMed: 23300705
DOI: 10.1371/journal.pone.0052549 -
Brain Research. Molecular Brain Research Mar 1994The human vesicular monoamine transporter (hSVMT) cDNA predicts a protein of 515 amino acids that shares 92% amino acid identity with the rat cDNA. Northern analyses...
The human vesicular monoamine transporter (hSVMT) cDNA predicts a protein of 515 amino acids that shares 92% amino acid identity with the rat cDNA. Northern analyses reveal expression of 4.3 kb SVMT mRNAs in rat hypothalamus, midbrain and brainstem, a 3 kb hSVMT mRNA in human brainstem and a 4.8 kb hSVMT mRNA in human hypothalamus. In situ hybridization documents significant SVMT expression in human nigra compacta neurons and in rat hypothalamic neurons whose distribution patterns are identical to those previously reported to display histaminergic markers. COS cell hSVMT expression yielded nanomolar affinities for tetrabenazine and reserpine, micromolar affinities for haloperidol, GBR12909, serotonin, mazindol, nomifensin and d-amphetamine, while dopamine, epinephrine, norepinephrine and histamine each displayed millimolar affinities. These observations extend the pharmacological characterization of hSVMT and studies of its distribution, and indicate likely physiological roles for SVMT in packaging monoamine transmitters including histamine.
Topics: Animals; Biological Transport; Brain Stem; Glycoproteins; Humans; In Situ Hybridization; Membrane Glycoproteins; Membrane Transport Proteins; Neuropeptides; Neurotransmitter Agents; RNA, Messenger; Radioligand Assay; Rats; Vesicular Biogenic Amine Transport Proteins; Vesicular Monoamine Transport Proteins
PubMed: 7912402
DOI: 10.1016/0169-328x(94)90050-7 -
Journal of Neurochemistry Jun 1999The formation of hydroxyl radicals following the systemic administration of 3,4-methylenedioxymethamphetamine (MDMA) was studied in the striatum of the rat by...
The formation of hydroxyl radicals following the systemic administration of 3,4-methylenedioxymethamphetamine (MDMA) was studied in the striatum of the rat by quantifying the stable adducts of salicylic acid and D-phenylalanine, namely, 2,3-dihydroxybenzoic acid (2,3-DHBA) and p-tyrosine, respectively. The repeated administration of MDMA produced a sustained increase in the extracellular concentration of 2,3-DHBA and p-tyrosine, as well as dopamine. The MDMA-induced increase in the extracellular concentration of both dopamine and 2,3-DHBA was suppressed in rats treated with mazindol, a dopamine uptake inhibitor. Mazindol also attenuated the long-term depletion of serotonin (5-HT) in the striatum produced by MDMA without altering the acute hyperthermic response to MDMA. These results are supportive of the view that MDMA produces a dopamine-dependent increase in the formation of hydroxyl radicals in the striatum that may contribute to the mechanism whereby MDMA produces a long-term depletion of brain 5-HT content.
Topics: Animals; Corpus Striatum; Hydroxybenzoates; Hydroxyl Radical; Kinetics; Male; Mazindol; Microdialysis; N-Methyl-3,4-methylenedioxyamphetamine; Phenylalanine; Rats; Rats, Sprague-Dawley; Salicylic Acid; Serotonin; Tyrosine
PubMed: 10349862
DOI: 10.1046/j.1471-4159.1999.0722516.x -
Virology Aug 1996Rats experimentally infected with the neurotropic RNA virus, Borna disease virus, have a hyperactive movement disorder. Because locomotor activity is modulated by the...
Rats experimentally infected with the neurotropic RNA virus, Borna disease virus, have a hyperactive movement disorder. Because locomotor activity is modulated by the nucleus accumbens (N. Acc.) dopamine (DA) system, high-affinity DA uptake, DA D1, D2, and D3 receptor binding sites were examined in N. Acc. subregions of normal and infected rats by quantitative receptor autoradiography. The N. Acc. of infected rats had decreased mazindol and D2 and D3 radioligand binding in the core and decreased D3 radioligand binding in rostral subregions. The abnormalities observed in the N. Acc. DA system of infected rats may offer insights into the potential viral pathogenesis of psychiatric conditions with a dopaminergic substrate such as schizophrenia and affective disorders.
Topics: Animals; Autoradiography; Borna Disease; Borna disease virus; Hyperkinesis; In Situ Hybridization; Male; Nucleus Accumbens; RNA, Viral; Rats; Rats, Inbred Lew; Receptors, Dopamine; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, Dopamine D3
PubMed: 8806517
DOI: 10.1006/viro.1996.0430 -
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyride neurotoxicity is attenuated in mice overexpressing Bcl-2.The Journal of Neuroscience : the... Oct 1998The proto-oncogene Bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that Bcl-2 protects against free radicals and that it increases...
The proto-oncogene Bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that Bcl-2 protects against free radicals and that it increases mitochondrial calcium-buffering capacity. The neurotoxicity of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyride (MPTP) is thought to involve both mitochondrial dysfunction and free radical generation. We therefore investigated MPTP neurotoxicity in both Bcl-2 overexpressing mice and littermate controls. MPTP-induced depletion of dopamine and loss of [3H]mazindol binding were significantly attenuated in Bcl-2 overexpressing mice. Protection was more profound with an acute dosing regimen than with daily MPTP administration over 5 d. 1-Methyl-4-phenylpyridinium (MPP+) levels after MPTP administration were similar in Bcl-2 overexpressing mice and littermates. Bcl-2 blocked MPP+-induced activation of caspases. MPTP-induced increases in free 3-nitrotyrosine levels were blocked in Bcl-2 overexpressing mice. These results indicate that Bcl-2 overexpression protects against MPTP neurotoxicity by mechanisms that may involve both antioxidant activity and inhibition of apoptotic pathways.
Topics: 1-Methyl-4-phenylpyridinium; 3,4-Dihydroxyphenylacetic Acid; Animals; Apoptosis; Brain Chemistry; Caspase 2; Caspase Inhibitors; Caspases; Dopamine; Dopamine Agents; Dopamine Uptake Inhibitors; Female; Free Radicals; Gene Expression; Homovanillic Acid; Injections, Intraperitoneal; MPTP Poisoning; Male; Mazindol; Mice; Mice, Transgenic; Neurons; Neurotoxins; Oxidative Stress; Pregnancy; Proto-Oncogene Proteins c-bcl-2; Radioligand Assay; Tritium; Tyrosine
PubMed: 9763461
DOI: 10.1523/JNEUROSCI.18-20-08145.1998 -
NPJ Schizophrenia Dec 2019The dopamine hypothesis proposes that there is a hypodopaminergic state in the prefrontal cortex and a hyperdopaminergic state in the striatum of patients with...
The dopamine hypothesis proposes that there is a hypodopaminergic state in the prefrontal cortex and a hyperdopaminergic state in the striatum of patients with schizophrenia. Evidence suggests the hyperdopaminergic state in the striatum is due to synaptic dopamine elevation, particularly in the dorsal striatum. However, the molecular mechanisms causing disrupted dopaminergic function in schizophrenia remains unclear. We postulated that the dopamine transporter (DAT), which regulates intra-synaptic dopamine concentrations by transporting dopamine from the synaptic cleft into the pre-synaptic neuron, could be involved in dopaminergic dysfunction in schizophrenia. Therefore, we measured levels of DAT in the cortex and striatum from patients with schizophrenia and controls using postmortem human brain tissue. Levels of desmethylimipramine-insensitive mazindol-sensitive [H]mazindol binding to DAT were measured using in situ radioligand binding and autoradiography in gray matter from Brodmann's area (BA) 10, BA 17, the dorsal striatum, and nucleus accumbens from 15 patients with schizophrenia and 15 controls. Levels of desmethylimipramine-insensitive mazindol-sensitive [H]mazindol binding were significantly higher in BA 10 from patients with schizophrenia (p = 0.004) and significantly lower in the dorsal striatum (dorsal putamen p = 0.005; dorsal caudate p = 0.007) from those with the disorder. There were no differences in levels of desmethylimipramine-insensitive [H]mazindol binding in BA 17 or nucleus accumbens. These data raise the possibility that high levels of DAT in BA 10 could be contributing to lower synaptic cortical dopamine, whereas lower levels of DAT could be contributing to a hyperdopaminergic state in the dorsal striatum.
PubMed: 31792225
DOI: 10.1038/s41537-019-0087-7 -
Journal of Nuclear Medicine : Official... Sep 2006The norepinephrine analog (11)C-meta-hydroxyephedrine (HED) is used with PET to map the regional distribution of cardiac sympathetic neurons. HED is rapidly transported...
UNLABELLED
The norepinephrine analog (11)C-meta-hydroxyephedrine (HED) is used with PET to map the regional distribution of cardiac sympathetic neurons. HED is rapidly transported into sympathetic neurons by the norepinephrine transporter (NET) and stored in vesicles. Although much is known about the neuronal mechanisms of HED uptake and retention, there is little information about the functional relationship between HED retention and cardiac sympathetic nerve density. The goal of this study was to characterize the dependence of HED retention on nerve density in rats with graded levels of cardiac denervation induced chemically with the neurotoxin 6-hydroxydopamine (6-OHDA).
METHODS
Thirty male Sprague-Dawley rats were divided into 6 groups, and each group was administered a different dose of 6-OHDA: 0 (controls), 7, 11, 15, 22, and 100 mg/kg intraperitoneally. One day after 6-OHDA injection, HED (3.7-8.3 MBq) was injected intravenously into each animal and HED concentrations in heart and blood at 30 min after injection were determined. Heart tissues were frozen and later processed by tissue homogenization and differential centrifugation into a membrane preparation for in vitro measurement of cardiac NET density. A saturation binding assay using (3)H-mazindol as the radioligand was used to measure NET density (maximum number of binding sites [B(max)], fmol/mg protein) for each heart.
RESULTS
In control animals, NET B(max) was 388 +/- 23 fmol/mg protein and HED heart uptake (HU) at 30 min was 2.89% +/- 0.35 %ID/g (%ID/g is percentage injected dose per gram tissue). The highest 6-OHDA dose of 100 mg/kg caused severe cardiac denervation, decreasing both NET B(max) and HED HU to 8% of their control values. Comparing values for all doses of 6-OHDA, HED retention had a strong linear correlation with NET density: HU = 0.0077B(max) -0.028, r(2) = 0.95.
CONCLUSION
HED retention is linearly dependent on NET density in rat hearts that have been chemically denervated with 6-OHDA, suggesting that HED retention is a good surrogate measure of NET density in the rat heart. This finding is discussed in relation to clinical observations of the dependence of HED retention on cardiac nerve density in human subjects using PET.
Topics: Animals; Carbon Radioisotopes; Ephedrine; Heart; Male; Metabolic Clearance Rate; Myocardium; Norepinephrine Plasma Membrane Transport Proteins; Radionuclide Imaging; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Sympathetic Nervous System; Tissue Distribution
PubMed: 16954558
DOI: No ID Found -
Biochemical Pharmacology Oct 2017Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake...
Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility strategies to identify the specific adduction site and binding pose for the crosslinkable, photoactive cocaine analog, RTI 82, which contains a photoactive azide attached at the 2β position of the tropane pharmacophore. Here, we utilize similar methodology with a different cocaine analog N-[4-(4-azido-3-I-iodophenyl)-butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane, MFZ 2-24, where the photoactive azide is attached to the tropane nitrogen. In contrast to RTI 82, which crosslinked into residue Phe319 of transmembrane domain (TM) 6, our findings show that MFZ 2-24 adducts to Leu80 in TM1 with modeling and biochemical data indicating that MFZ 2-24, like RTI 82, occupies the central S1 binding pocket with the (+)-charged tropane ring nitrogen coordinating with the (-)-charged carboxyl side chain of Asp79. The superimposition of the tropane ring in the three-dimensional binding poses of these two distinct ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site and the importance of the tropane moiety in competitive mechanisms of DA uptake inhibition. These findings set a structure-function baseline for comparison of typical and atypical DAT inhibitors and how their interactions with DAT could lead to the loss of cocaine-like behaviors.
Topics: Animals; Azides; Binding Sites; Cocaine; Cross-Linking Reagents; Dopamine Plasma Membrane Transport Proteins; Iodine Radioisotopes; LLC-PK1 Cells; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Structure; Mutant Proteins; Peptide Mapping; Photoaffinity Labels; Protein Binding; Structure-Activity Relationship; Substance-Related Disorders; Swine; Tropanes
PubMed: 28734777
DOI: 10.1016/j.bcp.2017.07.015 -
The Journal of Clinical Investigation Apr 1981Insulin-induced hypoglycemia by unknown mechanism(s) increases plasma arginine vasopressin (AVP) levels in humans. Mechanisms for increased AVP levels during central...
Insulin-induced hypoglycemia by unknown mechanism(s) increases plasma arginine vasopressin (AVP) levels in humans. Mechanisms for increased AVP levels during central nervous system glucoprivation were investigated by administering 20-min i.v. infusions of 2-deoxy-d-glucose (50 mg/kg), a competitive inhibitor of glucose utilization, or normal saline (sham), to 24 normal volunteers. Some of the infusions were administered in combination with neuropharmacological blocking agents (placebo). The behavioral, physiological, metabolic, and hormonal correlates of 2-deoxy-d-glucose (2DG)-induced gluco-privation and AVP secretion were studied in a group (n = 5) pretreated for 1 wk with either mazindol (1 mg per os three times per day), a potent norepinephrine and dopamine-reuptake blocker, or placebo. A second group (n = 5) received either propranolol (3 mg/3 min followed by 80 mug/min) or normal saline infusion before and during 2DG administration. With 2DG alone, plasma AVP levels increased from 1.3+/-0.3 pg/ml at base line to a peak of 4.5+/-1.4 pg/ml at 60 min and remained elevated for 150 min. From 30 to 180 min after 2DG administration, the 2DG-infused volunteers increased their water intake in comparison with sham-infused volunteers. Marked increases in epinephrine and slight increases in norepinephrine were associated with increases in plasma glucose and renin activity and decreases in plasma potassium. Plasma sodium and osmolality increased transiently and mean arterial pressure (MAP) fell. These changes, however, were small and inconstant and could not account for the observed increases in thirst and AVP levels. Pretreatment with mazindol prevented the decrease in MAP and the increase in plasma renin activity (PRA) following 2DG infusions without modifying increased thirst, water intake, or AVP responses to glucoprivation. Pretreatment with propranolol effectively blocked beta-adrenoreceptors as evidenced by increased MAP and plasma epinephrine, and abolition of the RPA increases during 2DG-induced glycoprivation, but did not suppress AVP and thirst responses. A cervical cord-sectioned patient lacking descending sympathetic out-flow had a potentiated thirst response to 2DG-induced glucoprivation in the absence of increases in sodium, catecholamines, and PRA. Thus 2DG administration activates mechanisms for increased thirst and AVP which are unrelated to changes in peripheral catecholamines, MAP, PRA, and osmolality.
Topics: Arginine Vasopressin; Central Nervous System; Deoxy Sugars; Deoxyglucose; Glucose; Humans; Male; Mazindol; Propranolol; Spinal Cord Injuries; Thirst
PubMed: 7204569
DOI: 10.1172/jci110121