Homology of a vesicular amine transporter to a gene conferring resistance to 1-methyl-4-phenylpyridinium.
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The vesicular amine transporter (VAT) catalyzes transport and storage of catechol and indolamines into subcellular organelles in a wide variety of cells. It plays a central role in neurotransmission and is the primary target for several pharmacological agents. One of the drugs, reserpine, binds very tightly to the transporter and remains bound even after solubilization, a finding that has proven useful for purification of the transporter from bovine adrenal medulla in a fully functional state. The sequences of 26 N-terminal amino acids and of an additional 7-amino acid internal peptide are presented. Antibodies against a synthetic peptide based on the above sequences immunoprecipitate the transporter, confirming the conclusion that the peptide sequence is derived from bovine VAT. To our knowledge, documentation of sequences of vesicular neurotransmitter transporters has not been presented previously. In addition, the sequences obtained are highly homologous to the predicted sequence of a protein from PC12 cells that confers to Chinese hamster ovary cells resistance to 1-methyl-4-phenylpyridinium (MPP+), an agent that causes parkinsonism in model systems, confirming the hypothesis that the protein conferring resistance to MPP+ is a VAT. (+info)
Expression cloning of a reserpine-sensitive vesicular monoamine transporter.
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A cDNA for a rat vesicular monoamine transporter, designated MAT, was isolated by expression cloning in a mammalian cell line (CV-1). The cDNA sequence predicts a protein of 515 amino acids with 12 putative membrane-spanning domains. The characteristics of [3H]serotonin accumulation by CV-1 cells expressing the cDNA clone suggested sequestration by an intracellular compartment. In cells permeabilized with digitonin, uptake was ATP dependent with an apparent Km of 1.3 microM. Uptake was abolished by the proton-translocating ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and with tri-(n-butyl)tin, an inhibitor of the vacuolar H(+)-ATPase. The rank order of potency to inhibit uptake was reserpine > tetrabenazine > serotonin > dopamine > norepinephrine > epinephrine. Direct comparison of [3H]monoamine uptake indicated that serotonin was the preferred substrate. Photolabeling of membranes prepared from CV-1 cells expressing MAT with 7-azido-8-[125I]iodoketanserin revealed a predominant tetrabenazine-sensitive photolabeled glycoprotein with an apparent molecular mass of approximately 75 kDa. The mRNA that encodes MAT was present specifically in monoamine-containing cells of the locus coeruleus, substantia nigra, and raphe nucleus of rat brain, each of which expresses a unique plasma membrane reuptake transporter. The MAT cDNA clone defines a vesicular monoamine transporter representing a distinct class of neurotransmitter transport molecules. (+info)
Uptake of meta-iodobenzylguanidine in neuroendocrine tumours is mediated by vesicular monoamine transporters.
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The radio-iodinated noradrenaline analogue meta-iodobenzylguanidine (MIBG) can be used for scintigraphy and radiation therapy of neuroendocrine (NE). The aim of the present study was to study the importance of vesicular monoamine transporters (VMATs) for the uptake of (123)I-MIBG in NE tumours. In nude mice, bearing the human transplantable midgut carcinoid GOT1, all organs and xenografted tumours accumulated (123)I after i.v. injection of (123)I-MIBG. A high concentration of (123)I was maintained in GOT1 tumours and adrenals, which expressed VMATs, but rapidly decreased in all other tissues. In the VMAT-expressing NE tumour cell lines GOT1 and BON and in VMAT-expressing primary NE tumour cell cultures (carcinoids, n=4 and pheochromocytomas, n=4), reserpine significantly reduced the uptake of (123)I-MIBG. The membrane pump inhibitor clomipramine had no effect on the uptake of (123)I-MIBG in GOT1 and BON cells, but inhibited the uptake in one out of four primary carcinoid cell cultures and three out of four primary pheochromocytoma cell cultures. In conclusion, VMATs and secretory granules are of importance for the uptake and retention of (123)I-MIBG in NE tumours. Information about the type and degree of expression of VMATs in NE tumours may be helpful in future to select patients suitable for radiation therapy with radio-iodinated MIBG. (+info)
Transforming growth factor-alpha directly augments histidine decarboxylase and vesicular monoamine transporter 2 production in rat enterochromaffin-like cells.
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For the production and vesicle storage of histamine, Enterochromaffin-like (ECL) cells express histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (VMAT2). Although HDC and VMAT2 show dynamic changes during gastric ulcer healing, the control system of their expression has not been fully investigated. In the present study, we investigated the effect of transforming growth factor-alpha (TGF-alpha) and proinflammatory cytokines on HDC and VMAT2 expression in rat ECL cells. Time course changes in the expression of TGF-alpha during the healing of acetic acid-induced ulcers were studied. EGF receptor (EGFR) expression was also examined in ECL cells, whereas the direct effects of TGF-alpha and proinflammatory cytokines on HDC and VMAT2 expression in ECL cells were investigated using in vivo and in vitro models. During the process of ulcer healing, expression of TGF-alpha mRNA was markedly augmented. Furthermore, EGFR was identified in isolated ECL cells. TGF-alpha stimulated HDC and VMAT2 mRNA expression and protein production and also increased histamine release from ECL cells. Selective EGFR tyrosine kinase inhibitor tyrphostin AG1478 almost completely inhibited HDC and VMAT2 gene expression induced by TGF-alpha in vivo and in vitro. During gastric mucosal injury, TGF-alpha was found to stimulate ECL cell functions by increasing HDC and VMAT2 expression. (+info)
Modification by dopaminergic drugs of choice behavior under concurrent schedules of intravenous saline and food delivery in monkeys.
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The allocation of "choice" behavior provides a measure that may be useful in developing experimental models of clinical relapse. In the present experiments, indirect monoaminergic agonists [cocaine, 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909), desipramine, and citalopram], and dopaminergic D1 family agonists [(+/-)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e (SKF 82958), R-(+)-6-bromo-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (R-(+)-6-BrAPB), and 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzaz epine (SKF 83959)] and D2 family agonists [quinelorane, R-(-)-10,11-dihydroxy-N-n-propylnorapomorphine (R-NPA), (+)-N-propyl-hydroxynaphoxazine [(+)-PHNO], and S-(+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4- oxazin-9-ol (PD 128907)] were evaluated for their capacity to alter the distribution of choice behavior in cocaine-experienced monkeys. Rhesus monkeys responded on two levers (injection-lever and food-lever) under concurrent fixed ratio 30; fixed ratio 30 schedules of i.v. cocaine and food delivery. Under training conditions, the distribution of behavior was related to the unit dose of i.v. cocaine: when saline was available, responding occurred predominantly on the food-lever and when reinforcing doses of cocaine were available, responding occurred predominantly on the injection-lever. Drugs were studied by administering i.m. pretreatment doses before components in sessions of i.v. saline availability. Cocaine produced dose-related increases in injection-lever responding in all monkeys, whereas desipramine failed to alter the distribution of behavior in any monkey. The dopamine transport blocker GBR 12909 and each dopamine D1 family agonist markedly increased injection-lever responding in three of four monkeys; the serotonin transport blocker citalopram and D2 family agonists were comparably effective in only one or two monkeys. These results agree with previous findings of similarity in the behavioral effects of cocaine and indirect or direct dopamine agonists and suggest, furthermore, that i.v. self-administration behavior engendered by priming doses of cocaine may involve actions mediated through both D1 and D2 families of dopamine receptors. (+info)
Cocaine-like discriminative stimulus effects of heroin: modulation by selective monoamine transport inhibitors.
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In previous studies, heroin was shown to engender cocaine-like discriminative stimulus (DS) effects; however, the mechanisms underlying the cocaine-like effects of heroin are unknown. The present study evaluated the extent to which the shared DS effects of heroin and cocaine involve common monoaminergic mechanisms of action. In squirrel monkeys discriminating cocaine (0.3 mg/kg) from saline, heroin engendered full or partial substitution for cocaine in three of four monkeys. Pretreatment with the selective dopamine transport inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909) dose dependently enhanced the cocaine-like DS effects of heroin in these three monkeys as well as the DS effects of cocaine in all subjects. Neither talsupram, a noradrenergic transport inhibitor, nor prazosin, a noradrenergic antagonist selective for alpha-1 receptors, systematically altered the cocaine-like DS effects of heroin at doses that enhanced (talsupram) or attenuated (prazosin) the DS effects of cocaine. Pretreatment with the serotonin uptake inhibitor citalopram similarly failed to alter the cocaine-like DS effects of heroin at doses that attenuated the DS effects of cocaine. Altogether, these findings suggest that heroin shares DS effects with cocaine in a subset of monkeys, and these cocaine-like effects are mediated at least in part by enhanced dopaminergic activity. Unlike the DS effects of cocaine itself, however, the cocaine-like DS effects of heroin do not appear to involve either noradrenergic or serotonergic mechanisms. (+info)
Phosphorylation and regulation of psychostimulant-sensitive neurotransmitter transporters.
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The neuronal transporters for the monoamines dopamine, serotonin, and norepinephrine are plasma membrane proteins that serve vital functions in the reuptake and control of synaptic neurotransmitter levels. They are also targets for abused and therapeutic drugs and play pivotal roles in neurological disorders such as depression, schizophrenia, and Parkinson's disease. There is increasing evidence that some activities of these carriers are subject to acute control by treatments that affect phosphorylation pathways, but the molecular basis for this is not understood. Recent work suggests that these regulatory processes may involve phosphorylation of the transporters by protein kinase C and other kinases, and may occur by affecting intrinsic transport activity or by controlling transporter cell surface expression. Phosphorylation-mediated regulation of monoamine transporters provides the potential for acute presynaptic control of neurotransmitter levels during normal neurophysiologic events, and dysregulation of these processes may lead to inappropriate transmitter clearance that contributes to the etiology of neurological disorders. (+info)
Effects of methamphetamine and lobeline on vesicular monoamine and dopamine transporter-mediated dopamine release in a cotransfected model system.
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Dopamine (DA) retention and drug-induced release kinetics were characterized in human embryonic kidney (HEK)-293 cells stably coexpressing the human DA transporter (hDAT) and human vesicular monoamine transporter (hVMAT2). Cofunction of hDAT and hVMAT2 caused greater retention of [3H]DA at 20 min (37 degrees C), or 45 min (22 degrees C) compared with cells that were treated with dihydrotetrabenazene (DHTB) to block the hVMAT2. In hDAT- and hVMAT2-coexpressing cells treated with DHTB during [3H]DA loading, methamphetamine (METH)-induced efflux was only 20% of preloaded [3H]DA, compared with 50 to 60% efflux in the absence of DHTB. Interestingly, the presence of DHTB (during release only) increased the potency and efficacy of METH at inducing [3H]DA release (without DHTB: EC50=33.8 microM, maximal release 51%; release with DHTB: EC50=3.2 microM, maximal release 61%), suggesting that the effects of METH and DHTB on vesicular storage are additive. High concentrations of lobeline induced a statistically significant release of [3H]DA from HEK-hDAT-hVMAT2 cells, but only in the absence of DHTB, suggesting an hVMAT2-mediated effect. Likewise, lobeline did not induce a significant release of [3H]DA from HEK-hDAT cells. The substrates DA and p-tyramine induced robust release of preloaded [3H]DA from cotransfected cells. Cocaine was somewhat effective at blocking substrate-induced [3H]DA efflux. These results suggest that coexpression of the hDAT and hVMAT2 can be used as a model system to distinguish functional pools of DA and to quantify differences in drug effects on DA disposition. In addition, cotransfected cells can be used to determine mechanisms of simultaneous drug interactions at multiple sites. (+info)