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Kathryn A Cunningham - One of the best experts on this subject based on the ideXlab platform.
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discriminative stimulus effects of ephedrine in rats analysis with Catecholamine Transporter and receptor ligands
Drug and Alcohol Dependence, 2003Co-Authors: Lance R Mcmahon, Kathryn A CunninghamAbstract:Abstract A drug discrimination procedure was used to examine the neuropharmacology of (−)-ephedrine (5 mg/kg), a sympathomimetic amine found in a variety of dietary supplements. (−)-Ephedrine has caused concern because of its use as a precursor in the manufacture of street drugs (e.g. methamphetamine) and its potential for abuse and toxicity. In the present study, the Catecholamine reuptake inhibitors mazindol and nomifensine, the norepinephrine (NE) reuptake inhibitor desipramine, and the dopamine D2-like (e.g. D2, D3 and D4) agonist quinpirole substituted for (−)-ephedrine (⩾80% (−)-ephedrine-lever responding). The NE reuptake inhibitor nisoxetine, the D1-like (e.g. D1 and D5) agonists (±)-SKF 38393 and SKF 82958, and the mixed D1-/D2-like agonist apomorphine occasioned intermediate levels of responding (50–79% (−)-ephedrine-lever responding). The (−)-ephedrine cue was antagonized by the D1-like antagonist SCH 23390 and the α1-adrenoceptor antagonist prazosin as well as the D2-like antagonists (−)-eticlopride and haloperidol, although only at doses that disrupted responding in some rats. The discriminative stimulus effects of a small dose of (−)-ephedrine (1.25 mg/kg) were enhanced by the α2-adrenoceptor antagonist idazoxan and to a lesser extent by the β-adrenoceptor antagonist (−)-propranolol. However, the α2-adrenoceptor agonist clonidine (0.04 mg/kg) did not attenuate the (−)-ephedrine stimulus. These results suggest that D1-, D2-like, and α1-adrenergic receptors mediate the discriminative stimulus effects of (−)-ephedrine. Substitution of desipramine for (−)-ephedrine and not for some other stimulants suggests that NE transmission is a prominent feature of the (−)-ephedrine discriminative stimulus, and that NE underlies therapeutic and abuse-related effects of (−)-ephedrine that diverge from those of other stimulants.
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Discriminative stimulus effects of (−)-ephedrine in rats: analysis with Catecholamine Transporter and receptor ligands
Drug and alcohol dependence, 2003Co-Authors: Lance R Mcmahon, Kathryn A CunninghamAbstract:Abstract A drug discrimination procedure was used to examine the neuropharmacology of (−)-ephedrine (5 mg/kg), a sympathomimetic amine found in a variety of dietary supplements. (−)-Ephedrine has caused concern because of its use as a precursor in the manufacture of street drugs (e.g. methamphetamine) and its potential for abuse and toxicity. In the present study, the Catecholamine reuptake inhibitors mazindol and nomifensine, the norepinephrine (NE) reuptake inhibitor desipramine, and the dopamine D2-like (e.g. D2, D3 and D4) agonist quinpirole substituted for (−)-ephedrine (⩾80% (−)-ephedrine-lever responding). The NE reuptake inhibitor nisoxetine, the D1-like (e.g. D1 and D5) agonists (±)-SKF 38393 and SKF 82958, and the mixed D1-/D2-like agonist apomorphine occasioned intermediate levels of responding (50–79% (−)-ephedrine-lever responding). The (−)-ephedrine cue was antagonized by the D1-like antagonist SCH 23390 and the α1-adrenoceptor antagonist prazosin as well as the D2-like antagonists (−)-eticlopride and haloperidol, although only at doses that disrupted responding in some rats. The discriminative stimulus effects of a small dose of (−)-ephedrine (1.25 mg/kg) were enhanced by the α2-adrenoceptor antagonist idazoxan and to a lesser extent by the β-adrenoceptor antagonist (−)-propranolol. However, the α2-adrenoceptor agonist clonidine (0.04 mg/kg) did not attenuate the (−)-ephedrine stimulus. These results suggest that D1-, D2-like, and α1-adrenergic receptors mediate the discriminative stimulus effects of (−)-ephedrine. Substitution of desipramine for (−)-ephedrine and not for some other stimulants suggests that NE transmission is a prominent feature of the (−)-ephedrine discriminative stimulus, and that NE underlies therapeutic and abuse-related effects of (−)-ephedrine that diverge from those of other stimulants.
Susan G. Amara - One of the best experts on this subject based on the ideXlab platform.
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The antidepressant-sensitive dopamine Transporter in Drosophila melanogaster: a primordial carrier for Catecholamines.
Molecular pharmacology, 2001Co-Authors: Peter Pörzgen, Sang Ki Park, Jay Hirsh, Mark S. Sonders, Susan G. AmaraAbstract:Extracellular concentrations of monoamine neurotransmitters are regulated by a family of high-affinity Transporters that are the molecular targets for such psychoactive drugs as cocaine, amphetamines, and therapeutic antidepressants. In Drosophila melanogaster, cocaine-induced behaviors show striking similarities to those induced in vertebrate animal models. Although a cocaine-sensitive serotonin carrier exists in flies, there has been no pharmacological or molecular evidence to support the presence of distinct carrier subtypes for other bioactive monoamines. Here we report the cloning and characterization of a cocaine-sensitive fly dopamine Transporter (dDAT). In situ hybridization demonstrates that dDAT mRNA expression is restricted to dopaminergic cells in the fly nervous system. The substrate selectivity of dDAT parallels that of the mammalian DATs in that dopamine and tyramine are the preferred substrates, whereas octopamine is transported less efficiently, and serotonin not at all. In contrast, dDAT inhibitors display a rank order of potency most closely resembling that of mammalian norepinephrine Transporters. Cocaine has a moderately high affinity to the cloned dDAT (IC50 = 2.6 μM). Voltage-clamp analysis of dDAT expressed in Xenopus laevis oocytes indicates that dDAT-mediated uptake is electrogenic; however, dDAT seems to lack the constitutive leak conductance that is characteristic of the mammalian Catecholamine Transporters. The combination of a DAT-like substrate selectivity and norepinephrine Transporter-like inhibitor pharmacology within a single carrier, and results from phylogenetic analyses, suggest that dDAT represents an ancestral Catecholamine Transporter gene. The identification of a cocaine-sensitive target linked to dopaminergic neurotransmission in D. melanogaster will serve as a basis for further dissection of the genetic components of psychostimulant-mediated behavior.
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Structural domains of Catecholamine Transporter chimeras involved in selective inhibition by antidepressants and psychomotor stimulants.
Molecular pharmacology, 1995Co-Authors: Kari J. Buck, Susan G. AmaraAbstract:Reuptake systems for monoamines are the initial sites of action for a wide range of therapeutic antidepressants and drugs of abuse, such as cocaine. To delineate structural domains of the dopamine and norepinephrine Transporters that contribute to differential interaction with reuptake inhibitors with antidepressant or reinforcing properties, a series of recombinant Transporter chimeras were generated and transiently expressed in HeLa cells. The inhibition constants (Ki values) for cocaine and a variety of selective transport inhibitors were determined for each chimera. Analyses of functional chimeras delineate a segment spanning transmembrane domains 5-7 of the norepinephrine Transporter of primary importance for high affinity binding of tricyclic and nontricyclic antidepressants (e.g., Ki < 20nM desipramine or nisoxetine). In contrast, all chimeras containing dopamine Transporter sequences from this region resemble the dopamine Transporter, which demonstrates higher affinity for psychomotor stimulants compared with antidepressants (e.g., Ki = 391 +/- 39 nM cocaine compared with 9365 +/- 1260 nM desipramine). A region including transmembrane domains 1-3 of the norepinephrine Transporter also contributes to the interaction of desipramine and nisoxetine, whereas the analogous region of the dopamine Transporter influences the affinity for piperazine derivatives (e.g., GBR12909 and LR1111) that are selective for the dopamine Transporter. These analyses provide a framework for identifying the precise structural determinants of monoamine Transporters involved in selective interactions with antidepressant and psychomotor stimulant reuptake inhibitors.
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Cell-type-specific expression of Catecholamine Transporters in the rat brain
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1994Co-Authors: Dominique Lorang, Susan G. Amara, Richard B. SimerlyAbstract:The dopamine Transporter (DAT) and norepinephrine Transporter (NET) terminate Catecholaminergic neurotransmission at synapses by high-affinity sodium-dependent reuptake into presynaptic terminals, and are the initial sites of action for drugs of abuse and antidepressants. In the present study, we used in situ hybridization combined with immunohistochemistry to study the distribution of DAT and NET mRNA in the adult rat brain. Cells were first immunolabeled with antisera directed against one of the Catecholamine-synthetic enzymes, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), or phenylethanolamine-N-methyltransferase (PNMT), in order to identify dopaminergic, noradrenergic, or epinephrine-containing cells. The immunolabeled cells were subsequently assayed for their ability to express Catecholamine Transporter mRNAs by in situ hybridization using either a rat DAT or NET cRNA probe. All dopaminergic cell groups of the mesencephalon contained high levels of DAT mRNA but only the A12 and A13 dopaminergic cell groups of the diencephalon appear to express detectable levels of DAT. All norepinephrine-containing cell bodies in the brainstem (locus coeruleus and lateral tegmentum) appear to express NET mRNA. In contrast, epinephrine-containing cell bodies of the brainstem do not appear to express NET mRNA, which raises the possibility that epinephrine may utilize a Transporter that is distinct from the other bioactive amines, or may act as an endocrine regulator that does not require rapid reuptake mechanisms. Moreover, the cell-type-specific expression of Catecholamine Transporters suggests that DAT and NET gene expression may be closely linked to cellular mechanisms that specify transmitter phenotype. The termination of neurotransmission is a critical component of neural signaling and depends on the rapid removal of neurotransmitters from the synaptic cleft. Pharmacological evidence indicates that the action of monoamines at the synapse is terminated predominantly by rapid reuptake into presynaptic nerve endings via neurotransmitter-specific, high-affinity, Na(+)-dependent membrane Transporter proteins. The cDNAs encoding distinct Transporter proteins for the monoamines dopamine, norepinephrine, and serotonin have been cloned, expressed, and characterized in a variety of heterologous systems (Blakely et al., 1991; Giros et al., 1991; Hoffman et al., 1991; Kilty et al., 1991; Pacholczyk et al., 1991; Shimada et al., 1991; Usdin et al., 1991). Although the monoamine Transporters share a high degree of sequence homology, they are distinguished by their monoamine substrate specificities and by their differential sensitivities to a wide spectrum of transport antagonists. For example, pharmacological agents that potently inhibit norepinephrine and serotonin transport, such as desmethylimipramine and citalopram, have little effect on the activity of the dopamine Transporter (Javitch et al., 1983).(ABSTRACT TRUNCATED AT 400 WORDS)
Matthias E. Liechti - One of the best experts on this subject based on the ideXlab platform.
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In vitro pharmacology of pipradrol derivatives, 3,4-methylenedioxypyrovalerone, and naphyrone (1145.3)
The FASEB Journal, 2014Co-Authors: Matthias E. Liechti, Linda D. Simmler, Anna Rickli, Marius C. HoenerAbstract:Objective: The aim of the study was to characterize the in vitro pharmacology of novel psychoactive substances (NPS) found in “ivory wave” including the methylphenidate-like pipradrol derivatives desoxypipradrol (2-diphenylmethylpiperidine, 2-DPMP) and diphenylprolinol (diphenyl-2-pyrrolidinemethanol, D2PM) as well as the pyrovalerone cathinones 3,4-methylenedioxypyrovalerone (MDPV) and naphyrone (bath salts). Methods: We assessed 3H-monoamine uptake and reverse transport in HEK 293 cells expressing the human serotonin-, norepinephrine-, or dopamine reuptake Transporters (SERT, NET, or DAT, respectively). Binding affinities at monoaminergic receptors were assessed using displacement of radioactive ligands. Results: D2PM and 2-DPMP were selective Catecholamine Transporter inhibitors without Transporter-mediated substrate-releasing properties similar to methylphenidate. 2-DPMP was an equally potent DAT/NET inhibitor to methylphenidate, while D2PM was less potent. Compared with classical stimulants, 2-DPMP w...
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Pharmacological profiles of aminoindanes, piperazines, and pipradrol derivatives
Biochemical pharmacology, 2014Co-Authors: Linda D. Simmler, Anna Rickli, York Schramm, Marius C. Hoener, Matthias E. LiechtiAbstract:Aminoindanes, piperazines, and pipradrol derivatives are novel psychoactive substances found in "Ecstasy" tablets as replacements for 3,4-methylenedioxymethamphetamine (MDMA) or substances sold as "ivory wave." The pharmacology of these MDMA- and methylphenidate-like substances is poorly known. We characterized the pharmacology of the aminoindanes 5,6-methylenedioxy-2-aminoindane (MDAI), 5-iodoaminoindane (5-IAI), and 2-aminoindane (2-AI), the piperazines meta-chlorophenylpiperazine (m-CPP), trifluoromethylphenylpiperazine (TFMPP), and 1-benzylpiperazine (BZP), and the pipradrol derivatives desoxypipradrol (2-diphenylmethylpiperidine [2-DPMP]), diphenylprolinol (diphenyl-2-pyrrolidinemethanol [D2PM]), and methylphenidate. We investigated norepinephrine (NE), dopamine (DA), and serotonin (5-hydroxytryptamine [5-HT]) uptake inhibition using human embryonic kidney 293 (HEK 293) cells that express the respective human monoamine Transporters (NET, DAT, and SERT). We also evaluated the drug-induced efflux of NE, DA, and 5-HT from monoamine-preloaded cells and the binding affinity to monoamine Transporters and receptors, including trace amine-associated receptor 1 (TAAR1). 5-IAI and MDAI preferentially inhibited the SERT and NET and released 5-HT. 2-AI interacted with the NET. BZP blocked the NET and released DA. m-CPP and TFMPP interacted with the SERT and serotonergic receptors. The pipradrol derivatives were potent and selective Catecholamine Transporter blockers without substrate releasing properties. BZP, D2PM, and 2-DPMP lacked serotonergic activity and TAAR1 binding, in contrast to the aminoindanes and phenylpiperazines. In summary, all of the substances were monoamine Transporter inhibitors, but marked differences were found in their DAT vs. SERT inhibition profiles, release properties, and receptor interactions. The pharmacological profiles of D2PM and 2-DPMP likely predict a high abuse liability.
Daniel Scherman - One of the best experts on this subject based on the ideXlab platform.
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regulation of the chromaffin granule Catecholamine Transporter in cultured bovine adrenal medullary cells stimulus biosynthesis coupling
Journal of Neurochemistry, 2006Co-Authors: Claire Desnos, Mariepierre Laran, Daniel SchermanAbstract:: The transsynaptic induction of the monoamine Transporter present on the membrane of chromaffin granules was studied in primary cultures of dissociated bovine adrenomedullary cells submitted to a chronic secretory stimulation. The amount of the vesicular monoamine Transporter was assayed by binding of the specific ligand [3H]-dihydrotetrabenazine. After several days of incubation in the presence of high potassium, the concentration of [3H]-dihydrotetrabenazine binding sites was increased by a 1.5–2.5 factor. This increase was smaller in the presence of the cholinergic agonist carbachol. The long-term inductions of the vesicular monoamine Transporter, of tyrosine hydroxylase, and of acetylcholinesterase were of similar magnitude. Under the same conditions, we found no variation in either the activities of other Catecholamine biosynthetic enzymes (dopamine β-hydroxylase and DOPA decarboxylase), or in metabolic enzymes such as lactate dehydrogenase and cytochrome c oxidase, and a decrease in the cellular content of chromogranin A and cytochrome b-561. The induction of the vesicular monoamine Transporter was inhibited by the calcium channel antagonists, fluspirilene and nifedipine, and was increased by the agonist Bay K 8644. It was abolished by cycloheximide and actinomycin D. These results indicate that calcium entry into chromaffin cells increases the synthesis of the vesicular monoamine Transporter, presumably by transcriptional activation. Elevation of intracellular cyclic AMP concentration or activation of protein kinase C also induced an increase in the expression of the vesicular monoamine Transporter. Our results confirm that components of storage vesicle membranes are differentially regulated in response to secretory stimulation, as are several cytosolic or intravesicular soluble proteins. Moreover, the up-regulation of the vesicular monoamine Transporter supports our previous hypothesis that Catecholamine uptake into storage vesicles might be, in the adrenal medulla, a rate-limiting step in the formation of mature secretion vesicles.
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Regulation of the chromaffin granule Catecholamine Transporter in cultured bovine adrenal medullary cells: stimulus-biosynthesis coupling.
Journal of Neurochemistry, 2006Co-Authors: Claire Desnos, Mariepierre Laran, Daniel SchermanAbstract:The transsynaptic induction of the monoamine Transporter present on the membrane of chromaffin granules was studied in primary cultures of dissociated bovine adrenomedullary cells submitted to a chronic secretory stimulation. The amount of the vesicular monoamine Transporter was assayed by binding of the specific ligand [3H]-dihydrotetrabenazine. After several days of incubation in the presence of high potassium, the concentration of [3H]-dihydrotetrabenazine binding sites was increased by a 1.5-2.5 factor. This increase was smaller in the presence of the cholinergic agonist carbachol. The long-term inductions of the vesicular monoamine Transporter, of tyrosine hydroxylase, and of acetylcholinesterase were of similar magnitude. Under the same conditions, we found no variation in either the activities of other Catecholamine biosynthetic enzymes (dopamine beta-hydroxylase and DOPA decarboxylase), or in metabolic enzymes such as lactate dehydrogenase and cytochrome c oxidase, and a decrease in the cellular content of chromogranin A and cytochrome b-561. The induction of the vesicular monoamine Transporter was inhibited by the calcium channel antagonists, fluspirilene and nifedipine, and was increased by the agonist Bay K 8644. It was abolished by cycloheximide and actinomycin D. These results indicate that calcium entry into chromaffin cells increases the synthesis of the vesicular monoamine Transporter, presumably by transcriptional activation. Elevation of intracellular cyclic AMP concentration or activation of protein kinase C also induced an increase in the expression of the vesicular monoamine Transporter. Our results confirm that components of storage vesicle membranes are differentially regulated in response to secretory stimulation, as are several cytosolic or intravesicular soluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Randy D. Blakely - One of the best experts on this subject based on the ideXlab platform.
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Structural diversity in the Catecholamine Transporter gene family: molecular cloning and characterization of an L-epinephrine Transporter from bullfrog sympathetic ganglia.
Advances in pharmacology (San Diego Calif.), 1998Co-Authors: Randy D. Blakely, Subramaniam ApparsundaramAbstract:Publisher Summary Molecular cloning studies have revealed that distinct gene products encode Catecholamine neurotransmitters dopamine (DA) and L-norepinephrine (NE) Transporters (DATs and NETs, respectively). Anatomical mapping studies reveal that dopaminergic neurons elaborate DATs, whereas noradrenergic neurons express NETs, though each carrier can efficiently transport both Catecholamines. Distinct gene products appear to have been evolved in concert with the structural diversification of the Catecholamines themselves. Interestingly, neurons in the rodent brain, which express the enzyme phenylethanolamine-N-methyltransferase (PNMT) and thus are presumed to synthesize epinephrine (Epi) from NE, exhibit little or no expression of DAT or NET. These findings suggest that synapses formed by PNMT-positive neurons either do not require rapid clearance to carry out efficient chemical signaling or elaborate a molecularly distinct Transporter. In this regard, Episynthesizing neurons in amphibians have been reported to express Catecholamine Transporters with a pharmacology similar to that displayed by NET, though a specific carrier has yet to be isolated and characterized. For this purpose mRNA is isolated and a cDNA library is prepared from sympathetic ganglia of the bullfrog, Ram catesbtuna . This study resulted in identification of frog ET (fET), an amphibian Catecholamine Transporter with enhanced efficiency for L-Epi transport, that reveals an evolutionary path for divergence between NETs and ETs and raises the question of whether the two Transporters coexist in a single species.
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The Caenorhabditis elegans Gene T23G5.5 Encodes an Antidepressant- and Cocaine-Sensitive Dopamine Transporter
Molecular pharmacology, 1998Co-Authors: L. D. Jayanthi, Subramaniam Apparsundaram, M. D. Malone, E. Ward, David M. Miller, M. Eppler, Randy D. BlakelyAbstract: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 ( K m = 1.2 μm) 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 andd-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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human norepinephrine Transporter biosynthetic studies using a site directed polyclonal antibody
Journal of Biological Chemistry, 1994Co-Authors: Haley E Melikian, John K Mcdonald, G Rudnick, Kimberly R Moore, Randy D. BlakelyAbstract: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.