Methamphetamine increases dopamine transporter higher molecular weight complex formation via a dopamine- and hyperthermia-associated mechanism

Abstract

Multiple high-dose administrations of methamphetamine (METH) both rapidly (within hours) decrease plasmalemmal dopamine (DA) uptake and cause long-term deficits in DA transporter (DAT) levels and other dopaminergic parameters persisting weeks to months in rat striatum. In contrast, either a single administration of METH or multiple administrations of methylenedioxymethamphetamine (MDMA) cause less of an acute reduction in DA uptake and little or no persistent dopaminergic deficits. The long-term dopaminergic deficits caused by METH have been suggested, in part, to involve the DAT. Hence, this study assessed the impact of METH and MDMA administration on the DAT protein per se. Results revealed that multiple administrations of METH promoted formation of higher molecular weight (>170 kDa) DAT-associated protein complexes 24-48 hr after treatment. This increase was attenuated by either preventing hyperthermia or pretreatment with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine; notably, each of these manipulations has also been demonstrated previously to prevent the persistent deficits in dopaminergic function caused by METH treatment. In contrast, either a single injection of METH or multiple injections of MDMA caused little or no formation of these DAT complexes. The addition of the reducing agent beta-mercaptoethanol to samples prepared from METH-treated rats diminished the intensity of these complexes. Taken together, these data are the first to demonstrate higher molecular weight DAT complex formation in vivo and that such formation can be altered by both pharmacological and physiological manipulations. The implications of this phenomenon with regard to the neurotoxic potential of these stimulants are discussed.

Figure 1.

METH-induced decreases in [³H]DA uptake were prevented by blocking hyperthermia. Rats received METH (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals) or saline (4 injections of 1 ml/kg s.c.; 2 hr intervals) and were exposed to 6 or 24°C ambient temperature. Rats were decapitated 1 or 24 hr after the last METH or saline administration, and [³H]DA uptake was assessed (A). Core rectal temperatures during the treatment are shown in B. Inverted arrows represent time points of METH or saline injection. *Values different from saline-treated controls; #value different from the METH-treated/24°C group (p ≤ 0.05).

Figure 7.

αMT pretreatment prevented the METH-induced higher molecular weight DAT complex formation (A) although hyperthermia was maintained (B). Rats were pretreated with either αMT (2 injections of 60 mg/kg, i.p.; 5 and 1 hr before the first METH injection) or saline (2 injections of 1 ml/kg, s.c.; 5 and 1 hr before the first METH injection). Subsequently, rats received METH (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals) or saline (4 injections of 1 ml/kg, s.c.; 2 hr intervals) and were decapitated 24 hr after treatment. Inverted arrows represent time points of METH or saline injection. *Values different from saline-treated controls (p ≤ 0.05). In this representative blot, two independent samples are shown for each treatment group.

Figure 9.

MDMA did not decrease [³H]DA uptake (A) but increased core body temperature (B). Rats received METH (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals), MDMA (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals) or saline (4 injections of 1 ml/kg, s.c.; 2 hr intervals) and were decapitated 24 hr after treatment. Inverted arrows represent time points of MDMA, METH, or saline injection. *Values different from saline-treated controls (p ≤ 0.05).

Figure 2.

Increases in higher molecular weight DAT complex formation by multiple administrations of METH were blocked by preventing hyperthermia. Rats were maintained in an ambient temperature of 24°C before treatment. Rats received METH (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals) or saline (4 injections of 1 ml/kg, s.c.; 2 hr intervals) and were exposed to 6 or 24°C ambient temperature for the duration of the experiment. Rats were killed 1 hr (A) or 24 hr (B) after the last METH administration. In these representative blots, two independent samples are shown for each treatment group.

Figure 3.

Multiple administrations of METH increased higher molecular weight DAT complex formation as evidenced using an antibody directed against the N terminus of the DAT. Rats received METH (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals) or saline (4 injections of 1 ml/kg, s.c.; 2 hr intervals) and were decapitated 6–72 hr later. In this representative blot, two independent samples are shown for each treatment group.

Figure 4.

Multiple administrations of METH increased higher molecular weight DAT complex formation as evidenced using an antibody directed against the C terminus of the DAT. Rats received METH (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals) or saline (4 injections of 1 ml/kg, s.c.; 2 hr intervals) and were decapitated 6–36 hr later. In this representative blot, two independent samples are shown for each treatment group.

Figure 5.

Multiple administrations of METH did not alter D2 receptor (A) or TH (B) complex formation. Rats received METH (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals) or saline (4 injections of 1 ml/kg, s.c.; 2 hr intervals) and were decapitated 24–36 hr later. In this representative blot, two independent samples are shown for each treatment group.

Figure 6.

The increase in METH-induced higher molecular weight DAT complex formation was attenuated by the addition of βME. Rats received METH (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals) or saline (4 injections of 1 ml/kg, s.c.; 2 hr intervals) and were decapitated 24 hr later. Samples were then split and prepared either in the presence or absence of 1.4 m βME. In this representative blot, two independent samples are shown for each treatment group.

Figure 8.

A single administration of METH did not increase higher molecular weight DAT complex formation at 1 and 24 hr (A) or 4 and 6 hr (B) after injection. Rats received METH (1 injection of 15 mg/kg, s.c.) or saline (1 injection of 1 ml/kg, s.c.) and were decapitated 1–24 hr after treatment. In this representative blot, two independent samples are shown for each treatment group.

Figure 10.

Multiple administrations of MDMA caused lesser increases in higher molecular weight DAT complex formation than METH. Rats received METH (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals), MDMA (4 injections of 7.5 mg/kg, s.c.; 2 hr intervals), or saline (4 injections of 1 ml/kg, s.c.; 2 hr intervals) and were decapitated 24 hr after treatment. In this representative blot, two independent samples are shown for each treatment group.