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1.
J Pharmacol Exp Ther ; 367(1): 119-128, 2018 10.
Article in English | MEDLINE | ID: mdl-30108161

ABSTRACT

The selective estrogen receptor modulator tamoxifen increases extracellular dopamine in vivo and acts as a neuroprotectant in models of dopamine neurotoxicity. We investigated the effect of tamoxifen on dopamine transporter (DAT)-mediated dopamine uptake, dopamine efflux, and [3H]WIN 35,428 [(-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane] binding in rat striatal tissue. Tamoxifen dose-dependently blocked dopamine uptake (54% reduction at 10 µM) and amphetamine-stimulated efflux (59% reduction at 10 µM) in synaptosomes. It also produced a small but significant reduction in [3H]WIN 35,428 binding in striatal membranes, indicating a weak interaction with the substrate binding site in the DAT. Biotinylation and cysteine accessibility studies indicated that tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner and does not affect surface expression of the DAT. Additional studies with mutant DAT constructs D476A and I159A suggested a direct interaction between tamoxifen and a secondary substrate binding site of the transporter. Locomotor studies revealed that tamoxifen attenuates amphetamine-stimulated hyperactivity in rats but has no depressant or stimulant activity in the absence of amphetamine. These results suggest a complex mechanism of action for tamoxifen as a regulator of the DAT. Due to its effectiveness against amphetamine actions and its central nervous system permeant activity, the tamoxifen structure represents an excellent starting point for a structure-based drug-design program to develop a pharmacological therapeutic for psychostimulant abuse.


Subject(s)
Dopamine Plasma Membrane Transport Proteins/metabolism , Dopamine Uptake Inhibitors/pharmacology , Dopamine/metabolism , Tamoxifen/pharmacology , Amphetamine/pharmacology , Animals , Binding Sites/drug effects , Cell Line , Central Nervous System Stimulants/pharmacology , Cocaine/analogs & derivatives , Cocaine/pharmacology , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Male , Rats , Swine , Synaptosomes/metabolism
2.
J Neurochem ; 141(1): 31-36, 2017 04.
Article in English | MEDLINE | ID: mdl-28075498

ABSTRACT

As one of the primary mechanisms by which dopamine signaling is regulated, the dopamine transporter (DAT) is an attractive pharmacological target for the treatment of diseases based in dopaminergic dysfunction. In this work we demonstrate for the first time that the commonly prescribed breast cancer therapeutic tamoxifen and its major metabolites, 4-hydroxytamoxifen and endoxifen, inhibit DAT function. Tamoxifen inhibits [3 H]dopamine uptake into human DAT (hDAT)-N2A cells via an uncompetitive or mixed mechanism. Endoxifen, an active metabolite of tamoxifen, asymmetrically inhibits DAT function in hDAT-N2A cells, showing a preference for the inhibition of amphetamine-stimulated dopamine efflux as compared to dopamine uptake. Importantly, we demonstrate that the effects of tamoxifen and its metabolites on the DAT occur independently of its activity as selective estrogen receptor modulators. This work suggests that tamoxifen is inhibiting DAT function through a previously unidentified mechanism.


Subject(s)
Dopamine Plasma Membrane Transport Proteins/antagonists & inhibitors , Dopamine Plasma Membrane Transport Proteins/physiology , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/physiology , Tamoxifen/metabolism , Tamoxifen/pharmacology , Animals , Cell Line, Tumor , Dose-Response Relationship, Drug , Estrogen Antagonists/metabolism , Estrogen Antagonists/pharmacology , Humans , Mice , Selective Estrogen Receptor Modulators/metabolism , Selective Estrogen Receptor Modulators/pharmacology
3.
J Chem Neuroanat ; 83-84: 50-58, 2017 Oct.
Article in English | MEDLINE | ID: mdl-27585851

ABSTRACT

Although best known as a selective estrogen receptor modulator (SERM), tamoxifen is a drug with a wide range of activities. Tamoxifen has demonstrated some efficacy has a therapeutic for bipolar mania and is believed to exert these effects through inhibition of protein kinase C (PKC). As the symptoms of amphetamine treatment in rodents are believed to mimic the symptoms of a manic episode, many of the preclinical studies for this indication have demonstrated that tamoxifen inhibits amphetamine action. The amphetamine-induced increase in extracellular dopamine which gives rise to the 'manic' effects is due to interaction of amphetamine with the dopamine transporter. We and others have demonstrated that PKC reduces amphetamine-induced reverse transport through the dopamine transporter. In this review, we will outline the actions of tamoxifen as a SERM and further detail another known action of tamoxifen-inhibition of PKC. We will summarize the literature showing how tamoxifen affects amphetamine action. Finally, we will present our hypothesis that tamoxifen, or an analog, could be used therapeutically to reduce amphetamine abuse in addition to treating mania.


Subject(s)
Amphetamine-Related Disorders/drug therapy , Protein Kinase Inhibitors/pharmacology , Tamoxifen/pharmacology , Animals , Humans , Protein Kinase C/metabolism
4.
Am J Physiol Heart Circ Physiol ; 310(11): H1790-800, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27199131

ABSTRACT

Dysregulated nitric oxide (NO) signaling contributes to the pathogenesis of hypertension, a prevalent and often sex-specific risk factor for cardiovascular disease. We previously reported that mice deficient in the α1-subunit of the NO receptor soluble guanylate cyclase (sGCα1 (-/-) mice) display sex- and strain-specific hypertension: male but not female sGCα1 (-/-) mice are hypertensive on an 129S6 (S6) but not a C57BL6/J (B6) background. We aimed to uncover the genetic and molecular basis of the observed sex- and strain-specific blood pressure phenotype. Via linkage analysis, we identified a suggestive quantitative trait locus associated with elevated blood pressure in male sGCα1 (-/-)S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE). Renal expression of Cyp4a12a in mice was associated with genetic background, sex, and testosterone levels. In addition, 20-HETE levels were higher in renal preglomerular microvessels of male sGCα1 (-/-)S6 than of male sGCα1 (-/-)B6 mice. Furthermore, treating male sGCα1 (-/-)S6 mice with the 20-HETE antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) lowered blood pressure. Finally, 20-HEDE rescued the genetic background- and testosterone-dependent impairment of acetylcholine-induced relaxation in renal interlobar arteries associated with sGCα1 deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGCα1 deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling.


Subject(s)
Androgens/pharmacology , Cytochrome P450 Family 4/genetics , Hydroxyeicosatetraenoic Acids/metabolism , Hypertension/metabolism , Soluble Guanylyl Cyclase/metabolism , Animals , Blood Pressure/drug effects , Blood Pressure/physiology , Female , Genetic Linkage , Hypertension/genetics , Kidney/drug effects , Kidney/metabolism , Male , Mice , Mice, Knockout , Quantitative Trait Loci , Sex Factors , Soluble Guanylyl Cyclase/genetics , Testosterone/blood
5.
ACS Chem Neurosci ; 7(6): 757-66, 2016 06 15.
Article in English | MEDLINE | ID: mdl-26996926

ABSTRACT

Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate for the dopamine transporter and reverses the transporter to cause an increase in extracellular dopamine. Activation of the beta subunit of protein kinase C (PKCß) enhances extracellular dopamine in the presence of amphetamine by facilitating the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously demonstrated that PKCß inhibitors block amphetamine-stimulated dopamine efflux in synaptosomes from rat striatum in vitro. In this study, we utilized in vivo microdialysis in live, behaving rats to assess the effect of the PKCß inhibitors, enzastaurin and ruboxistaurin, on amphetamine-stimulated locomotion and increases in monoamines and their metabolites. A 30 min perfusion of the nucleus accumbens core with 1 µM enzastaurin or 1 µM ruboxistaurin reduced efflux of dopamine and its metabolite 3-methoxytyramine induced by amphetamine by approximately 50%. The inhibitors also significantly reduced amphetamine-stimulated extracellular levels of norepinephrine. The stimulation of locomotor behavior by amphetamine, measured simultaneously with the analytes, was comparably reduced by the PKCß inhibitors. Using a stable isotope label retrodialysis procedure, we determined that ruboxistaurin had no effect on basal levels of dopamine, norepinephrine, glutamate, or GABA. In addition, normal uptake function through the dopamine transporter was unaltered by the PKCß inhibitors, as measured in rat synaptosomes. Our results support the utility of using PKCß inhibitors to reduce the effects of amphetamine.


Subject(s)
Amphetamine/pharmacology , Corpus Striatum/drug effects , Dopamine Plasma Membrane Transport Proteins/drug effects , Dopamine/analogs & derivatives , Protein Kinase C beta/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Animals , Autoreceptors/metabolism , Corpus Striatum/metabolism , Dopamine/metabolism , Dopamine/pharmacology , Dopamine Plasma Membrane Transport Proteins/metabolism , Dopamine Uptake Inhibitors/pharmacology , Male , Microdialysis/methods , Neostriatum/drug effects , Neostriatum/metabolism , Rats, Sprague-Dawley
6.
J Neurochem ; 128(1): 152-61, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23889359

ABSTRACT

The ventrolateral thalamus (VL) is a primary relay point between the basal ganglia and the primary motor cortex (M1). Using dual probe microdialysis and locomotor behavior monitoring, we investigated the contribution of VL input into M1 during amphetamine (AMPH)-stimulated monoamine release and hyperlocomotion in rats. Tetrodotoxin (10 µM) perfusion into the VL significantly lowered hyperactivity induced by AMPH (1 mg/kg i.p.). This behavioral response corresponded to reduced cortical glutamate and monoamine release. To determine which glutamate receptors the thalamocortical projections acted upon, we perfused either the α-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)/kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) (10 µM) or the N-methyl-D-aspartic acid (NMDA) receptor antagonist (MK-801) intracortically followed by systemic AMPH. The results show that AMPA/kainate, and to a lesser extent NMDA receptors, mediated the observed effects. As glutamate-monoamine interactions could possibly occur through local or circuit-based mechanisms, we isolated and perfused M1 tissue ex vivo to determine the extent of local glutamate-dopamine interactions. Taken together, these results demonstrate that AMPH generates hyperlocomotive states via thalamocortical signaling and that cortical AMPA receptors are an important mediator of these effects. This study utilizes dual probe microdialysis sampling and comprehensive LC-MS analysis to determine the effects of amphetamine (1 mg/kg i.p.) on thalamocortical neurotransmission. Using pharmacological tools such as local thalamic tetrodotoxin (TTX) perfusion and glutamate antagonist at the cortical level, we demonstrate that thalamocortical glutamate (acting primarily through cortical AMPA receptors) is an essential component in amphetamine-induced hyperlocomotion.


Subject(s)
Amphetamine/pharmacology , Glutamic Acid/metabolism , Motor Activity/drug effects , Motor Activity/physiology , Motor Cortex/metabolism , Thalamus/metabolism , Animals , Male , Microdialysis/methods , Motor Cortex/drug effects , Rats , Rats, Sprague-Dawley , Thalamus/drug effects
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