Amphetamine and Methamphetamine Differentially Affect Dopamine Transporters in Vitro and in Vivo

Abstract

The psychostimulants D-amphetamine (AMPH) and methamphetamine (METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic neurons. Abnormal DA release is thought to occur by reverse transport through the DA transporter (DAT), and it is believed to underlie the severe behavioral effects of these drugs. Here we compare structurally similar AMPH and METH on DAT function in a heterologous expression system and in an animal model. In the in vitro expression system, DAT-mediated whole-cell currents were greater for METH stimulation than for AMPH. The dopamine transporter (DAT)3 is a main target for psychostimulants, such as D-amphetamine (AMPH), methamphetamine (METH), cocaine (COC), and methylphenidate (Ritalin). DAT is the major clearance mechanism for synaptic dopamine (DA) (1) and thereby regulates the strength and duration of dopaminergic signaling. AMPH and METH are substrates for DAT and competitively inhibit DA uptake (2, 3) and release DA through reverse transport (4 -9). AMPH-and METH-induced elevations in extracellular DA result in complex neurochemical changes and profound psychiatric effects (2, 10 -16). Despite their structural and pharmacokinetic similarities, a recent National Institute on Drug Abuse report describes METH as a more potent stimulant than AMPH with longer lasting effects at comparable doses (17). Although the route of METH administration and its availability must contribute to the almost four times higher lifetime nonmedical use of METH compared with AMPH (18), there may also be differences in the mechanisms that underlie the actions of these two drugs on the dopamine transporter.Recent studies by Joyce et al. (19) have shown that compared with D-AMPH alone, the combination of D-and L-AMPH in Adderall significantly prolonged the time course of extracellular DA in vivo. These experiments demonstrate that subtle structural features of AMPH, such as chirality, can affect its action on dopamine transporters. Here we investigate whether METH, a more lipophilic analog of AMPH, affects DAT differently than AMPH, particularly in regard to stimulated DA efflux.METH and AMPH have been reported as equally effective in increasing extracellular DA levels in rodent dorsal striatum (dSTR), nucleus accumbens (NAc) (10,14,20), striatal synaptosomes, and DAT-expressing cells in vitro (3, 6). John and

Intracellular Methamphetamine Prevents the Dopamine-induced Enhancement of Neuronal Firing

“…Recently, we demonstrated at equipotent concentrations, methamphetamine evokes a greater dopamine efflux via DAT than amphetamine and at a lower membrane potential (29). This greater effect of methamphetamine on DAT function was supported by in vivo chronomicroamperometry experiments in the nucleus accumbens (29). These findings are consistent with previous reports suggesting that methamphetamine-induced release of [ 3 H]dopamine from pre-loaded COS-7-DAT cells is significantly higher than that induced by amphetamine (23).…”

“…Our recent in vitro and in vivo studies reveal methamphetamine is not just a "strongeramphetamine"; it affects DAT differently. Recently, we demonstrated at equipotent concentrations, methamphetamine evokes a greater dopamine efflux via DAT than amphetamine and at a lower membrane potential (29). This greater effect of methamphetamine on DAT function was supported by in vivo chronomicroamperometry experiments in the nucleus accumbens (29).…”

“…Only GBR12935-sensitive neurons were included in the analysis. We and others have shown that after whole-cell patch clamp the pipette solution reaches equilibrium with the intracellular milieu within 5-8 min when the pipette resistance is ϳ4 -5 megaohms (29,36,40,41). To examine the consequences of intracellular amphetamine and methamphetamine on DAT-mediated current or changes in the neuronal firing rate, the base-line current or voltage recordings were acquired immediately after obtaining the whole cell configuration and 8 min after the initiation of whole-cell patch clamp.…”

“…Cell Lines-Chinese Hamster Ovary (CHO) and human embryonic kidney 293 EM4 cells (HEK) overexpressing human dopamine transporter tagged with yellow fluorescent protein (YFP) used in evaluating DAT-mediated currents were generous gifts from Dr. Jonathan Javitch, Columbia University (23,29). The N terminus of synthetic human DAT cDNA was fused with the C terminus of the coding region of enhanced YFP from pEYFP-N1 (Clontech) to generate a fusion construct denoted as YFP-DAT, which was then stably overexpressed in CHO and HEK cells (36).…”

Dopamine Transporter Activity Is Modulated by α-Synuclein

“…As predicted, KCl induces a large and rapid membrane depolarization (Fig. 5B) (47). Therefore, as compared with the AMPH treatment, KCl recruits a greater level of ␣-synuclein to the membrane even in the absence of DAT expression due to its robust increase in membrane depolarization.…”