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

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. At the same voltage and concentration, METH released five times more DA than AMPH and did so at physiological membrane potentials. At maximally effective concentrations, METH released twice as much Ca²⁺]_i from internal stores compared with AMPH. Ca²⁺]_i responses to both drugs were independent of membrane voltage but inhibited by DAT antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were required for the AMPH- and METH-induced increase in Ca²⁺]_i and for the enhanced effects of METH on Ca²⁺]_i elevation. Calmodulin-dependent protein kinase II and protein kinase C inhibitors alone or in combination also blocked AMPH- or METH-induced Ca²⁺ responses. Finally, in the rat nucleus accumbens, in vivo voltammetry showed that systemic application of METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting in excess external DA. Together these data demonstrate that METH has a stronger effect on DAT-mediated cell physiology than AMPH, which may contribute to the euphoric and addictive properties of METH compared with 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 Jones (21), however, have recently shown in mouse striatal and substantia nigra slices, that AMPH is a more potent inhibitor of DA uptake than METH. On the other hand, in synaptosomes METH inhibits DA uptake three times more effectively than AMPH (14), and in DAT-expressing COS-7 cells, METH releases DA more potently than AMPH (EC₅₀ = 0.2 μm for METH versus EC₅₀ = 1.7 μm for AMPH) (5). However, these differences do not hold up under all conditions. For example, in a study utilizing C6 cells, the disparity between AMPH and METH was not found (12).The variations in AMPH and METH data extend to animal models. AMPH- and METH-mediated behavior has been reported as similar (22), lower (20), or higher (23) for AMPH compared with METH. Furthermore, although the maximal locomotor activation response was less for METH than for AMPH at a lower dose (2 mg/kg, intraperitoneal), both drugs decreased locomotor activity at a higher dose (4 mg/kg) (20). In contrast, in the presence of a salient stimuli, METH is more potent in increasing the overall magnitude of locomotor activity in rats yet is equipotent with AMPH in the absence of these stimuli (23).The simultaneous regulation of DA uptake and efflux by DAT substrates such as AMPH and METH, as well as the voltage dependence of DAT (24), may confound the interpretation of existing data describing the action of these drugs. Our biophysical approaches allowed us to significantly decrease the contribution of DA uptake and more accurately determine DAT-mediated DA efflux with millisecond time resolution. We have thus exploited time-resolved, whole-cell voltage clamp in combination with in vitro and in vivo microamperometry and Ca²⁺ imaging to compare the impact of METH and AMPH on DAT function and determine the consequence of these interactions on cell physiology.We find that near the resting potential, METH is more effective than AMPH in stimulating DAT to release DA. In addition, at efficacious concentrations METH generates more current, greater DA efflux, and higher Ca²⁺ release from internal stores than AMPH. Both METH-induced or the lesser AMPH-induced increase in intracellular Ca²⁺ are independent of membrane potential. The additional Ca²⁺ response induced by METH requires intact phosphorylation sites in the N-terminal domain of DAT. Finally, our in vivo voltammetry data indicate that METH inhibits clearance of locally applied DA more effectively than AMPH in the rat nucleus accumbens, which plays an important role in reward and addiction, but not in the dorsal striatum, which is involved in a variety of cognitive functions. Taken together these data imply that AMPH and METH have distinguishable effects on DAT that can be shown both at the molecular level and in vivo, and are likely to be implicated in the relative euphoric and addictive properties of these two psychostimulants.