Regulation of the dopamine transporter - Addiction Research ...

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Schmitt & Reith DAT Regulation and that this trafficking can be rapidly regulated, findings of in vitro amphetamine-induced reduction in transporter radioligand binding were interpreted as evidence of the in vivo neurotoxicity of these compounds. With the new knowledge on DAT trafficking phenomena, such findings will need to be reinterpreted. In the following, we will discuss the possible implications of transporter protein regulation to the putative toxicity of several substituted amphetamine derivatives that are often encountered as drugs of addiction (the prototypical example being methamphetamine) and/or recreational drugs. We will also discuss the potential utility of agents that reduce plasmalemmal DAT expression in the rectification of dopaminergic neuroadaptation that occurs in chronic cocaine addiction. 37,40 The amphetamines are an immensely diverse class of compounds and the structure-activity relationship of the amphetaminergic structural framework has been thoroughly investigated. 112 Amphetamine derivatives can elicit a wide range of phenomenological and neurochemical effects; however, for this discussion, we will focus solely on stimulant-like amphetamine derivatives that act as monoamine transporter substrates and will not consider any of the psychedelic amphetamine derivatives, which act primarily at serotonin 5-HT2 receptors. 113 Whereas amphetamine itself exhibits roughly equipotent substrate activity at the DAT and NET with little meaningful activity at the SERT—amphetamine inhibits [ 3 H]DA and [ 3 H]NE uptake with 112- and 98fold greater potency than [ 3 H]5-HT, respectively— certain amphetamine analogues bearing substitutions on the phenyl ring and amine nitrogen have unique activity profiles at the three monoamine transporters. 114 For example, aromatic substitution with a bulky, electron-rich moiety (such as a methoxy group or a halogen larger than fluorine) at the 3- or 4-position usually decreases substrate activity at the DAT but concomitantly increases affinity toward the SERT. 115,116 Hence, amphetamine derivatives, such as 4-chloroamphetamine (PCA), 4-methoxyamphetamine (4-MA; more traditionally known as PMA, but we shall refer to it as 4-MA to prevent confusion with the phorbol ester �- PMA) and 3-trifluoromethyl-N-ethylamphetamine (fenfluramine) are often labeled as “serotonergic amphetamines” (although PCA still possesses significant affinity for both the DAT and NET 116 ). Alkylation of the amphetamine nitrogen to the secondary amine generally increases SERT affinity without drastically altering potency at either the DAT or NET; however, alkyl moieties longer than N-ethyl impede substratelike activity, with d-N-butylamphetamine being virtually inactive in behavioral assays. 117 Therefore, d-methamphetamine exhibits roughly 2.5-fold greater potency than d-amphetamine as a SERT substrate. 115 The empathogen 3,4methylenedioxyamphetamine (MDA) and its Nmethyl analogue (MDMA)—both widely used recreational drugs, often sold (with any number of admixes) under the name “ecstasy”—have affinity for all three of the monoamine transporters, with slightly greater substrate activity at the SERT and NET than at the DAT. 118–120 SERT-affecting substituted amphetamine analogues decrease plasmalemmal SERT expression, paralleling the endocytic effect that amphetamine itself has on the DAT. Acute administration of high doses of d-fenfluramine or PCA to rats decreases the Bmax for SERT radioligand binding by 30–60% (depending upon the interval after drug administration), without significant changes in whole-cell SERT protein levels, 121 as measured by Western blot analysis (but also see Xie et al. 122 ). MDMA has also been frequently shown to cause loss of both radiolabeled SERT and DAT binding sites in experimental animals. 123–125 Because monoaminergic neurotransmission is so intimately involved in cognition, affect, behavioral reinforcement, and motor function, 2,126 it is not surprising that the implications of substrate-induced monoamine transporter downregulation on human physiological and psychical function are highly debated topics. The amphetaminergic substrate that has received the most attention in this debate is MDMA. Because of its widespread popularity as a recreational drug and nearly ubiquitous presence in the party scene, ascertaining the potential of recreational doses of MDMA to elicit neurotoxicity in humans is an important public health question. Furthermore, elucidation of the mechanism underlying MDMAergic neurotoxicity will also provide insight into strategies to minimize potential damage dealt by recreational MDMA use. Initial neurochemical studies in rats revealed a dramatic and protracted reduction in the density of SERT expression and in total brain 5-HT content after high doses of either MDA or MDMA. 123 Similar losses of SERT expression were observed after Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences. 329
DAT Regulation Schmitt & Reith treatment with the well-established serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). Hence, authors of these early studies concluded that MDA-like drugs produce neurotoxic insult to serotonergic neurons and suggested that the magnitude and duration of the decline in SERT expression constitutes a method of quantifying the extent of this damage. The reduction in SERT expression that occurs after MDMA administration is quantitatively similar to reductions caused by 4-substituted amphetamines, such as 4-MA and PCA. 127,128 In light of the fact that membrane trafficking of monoamine transporter proteins is altered upon exposure to amphetaminergic substrates— and that such changes persist beyond the duration of acute drug exposure—the question is thus whether or not reductions in SERT expression represent mere changes in transporter trafficking and membrane distribution or true damage to serotonergic neuronal function. Evidence in support of both conclusions exists. Experiments by Wang et al. 125,129 showed that MDMA administration reduces radioligand binding to plasmalemmal SERTs in rats; but unlike the specific serotonergic toxin 5,7-DHT, it neither alters total SERT protein expression nor induces microglial activation. In contrast, the results of Xie et al. 122 —who used a different, more specific antibody for immunochemical detection of the SERT protein— do indicate substantial loss of SERT protein after treatment with either 5,7-DHT or the serotonergic amphetamines MDMA, PCA, and fenfluramine, suggesting functional damage to serotonergic nerve terminals (Ref. 122, Figs. 8–10). However, some of the differences seen by these two groups may be related to respective MDMA dosing regimens: 22.5 mg/kg total MDMA (3 × 7.5 mg/kg, each 2 h apart) was given in the studies by Wang et al., 125,129 whereas a total dose of 45 mg/kg (3 × 15 mg/kg, each 1.5 h apart) was used in the Xie et al. study. 122 Callaghan et al. compared the effects of repeated administration of either MDMA or 4-MA on in vitro measures of neurodegeneration (such as plasmalemmal SERT binding) with in vivo assessment of 5-HT clearance by using real-time microamperometry. 128 Here, the authors found that [ 3 H]cyanoimipramine binding to the SERT was significantly reduced 2 weeks after repeated administration of either high-dose 4-MA or MDMA (15 mg/kg, twice daily for 4 days); however, clearance of locally applied 5-HT was reduced only in rats treated with a high dose of 4-MA. The observation that 5-HT clearance in vivo was unaltered by even a high-dose MDMA treatment regimen suggests that in vitro measures, such as plasmalemmal SERT radioligand binding, do not necessarily predict the functional state of serotonergic neurons in vivo. Moreover, these findings highlight that the toxicity of substituted amphetamines can vary greatly depending upon their individual chemical structure. Here 4-MA appears to have a greater potential than MDMA to elicit toxicity in vivo. This finding is certainly consistent with epidemiological data, 130 which show a high incidence of mortality after consumption of clandestine pressed pills containing 4- MA sold as “ecstasy.” By contrast, fatalities caused by consumption of moderate doses of unadulterated MDMA (as opposed to ecstasy pills) in the absence of other drugs are exceedingly rare. Paradoxically, localized direct administration of MDMA in rats by in vivo reverse microdialysis— at concentrations equivalent to those attained after high-dose systemic administration—does not result in the typical indicators of serotonergic neurotoxicity, despite eliciting dramatically greater increases in extracellular monoamine levels. 131 Surprisingly, MDMA was demonstrated to exert neuroprotective effects in cultured rat fetal neurons, indicating that direct exposure to the chemical itself is not necessarily cytotoxic. 132 In addition, whereas systemically administered MDA and MDMA acutely increase the concentration of radical reactive oxygen species (ROS) and produce oxidative stress in the brain, direct intracerebroventricular administration is devoid of such effects. 131,133 The difference in toxicological profile between direct microdialysis and systemic routes of administration suggests that much of the toxicity of MDA and its N-alkylated cousins is the result of metabolic transformation of parent compounds into more potent neurotoxins. 134 In humans, MDA and MDMA are largely metabolized via demethylenation by the hepatic CYP2D6 enzyme to the respective 3,4-dihydroxyamphetamine species (�-methyldopamine and �,Ndimethyldopamine). 135 In recent years, several investigators have revealed that—much like dopamine—these catechol species readily undergo oxidation to semiquinone and quinone species that can form neurotoxic thioether compounds 330 Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences.
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