Methamphetamine-induced rapid and reversible reduction in the activities of tryptophan hydroxylase and dopamine transporters: oxidative consequences?

Treatment with high doses of methamphetamine (METH) results in dramatic changes in extrapyramidal monoaminergic systems. Elevated concentrations of extracellular dopamine (DA), caused by METH administration, are thought to contribute to these effects due to the oxidative potential of this reactive catecholamine. According to this hypothesis monoaminergic cellular elements, which are vulnerable to oxidative modification, may be especially sensitive to high-dose METH treatments. We confirmed this possibility by observing that both tryptophan hydroxylase (the synthesizing enzyme for serotonin) and the DA transporter, proteins particularly susceptible to oxidative modification, were rapidly (within 30 min), but reversibly (returned to control levels by 36 hr) inactivated by a single administration of METH. These findings suggest that there also may be other cellular elements similarly altered by METH treatment due to oxidative mechanisms.

Methamphetamine treatment rapidly inhibits serotonin, but not glutamate, transporters in rat brain.

Previous studies have demonstrated that multiple methamphetamine (METH) administrations rapidly and reversibly decrease dopamine transporter activity assessed in striatal synaptosomes. A role for reactive oxygen species was suggested by findings that: (1) METH treatment increases the formation of oxygen radicals in vivo; and (2) oxygen radicals, generated by the enzyme xanthine oxidase, attenuate dopamine uptake in vitro. To test the selectivity of transporter responses, the present study examined effects of METH and xanthine oxidase on [3H]serotonin ([3H]5HT) and [3H]glutamate transport into striatal synaptosomes. Multiple doses of METH, or incubation with xanthine oxidase, rapidly attenuated [3H]5HT transport; an effect attributable to a decrease in Vmax. The METH-induced decrease in transport activity completely recovered by 24 h, but was decreased again 1 week later. In contrast, [3H]glutamate transport was essentially unchanged after METH treatment or incubation with xanthine oxidase. These findings indicate that: (1) METH causes a rapid and reversible decrease in 5HT transporter activity; and (2) glutamate transporters are less susceptible than 5HT transporters to effects of reactive species or METH treatment.

Nature of methamphetamine-induced rapid and reversible changes in dopamine transporters.

The nature of methamphetamine-induced rapid and transient decreases in dopamine transporter activity was investigated. Regional specificity was demonstrated, since [3H]dopamine uptake was decreased in synaptosomes prepared from the striatum, but not nucleus accumbens, of methamphetamine-treated rats. Differences among effects on dopamine transporter activity and ligand binding were also observed, since a single methamphetamine administration decreased [3H]dopamine uptake without altering [3H]WIN35428 ([3H](-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate) binding in synaptosomes prepared 1 h after injection. Moreover, multiple methamphetamine injections caused a greater decrease in [3H]dopamine uptake than [3H]WIN35428 binding in synaptosomes prepared I h after dosing. Finally, decreases in [3H]dopamine uptake, but not [3H]WIN35428 binding, were partially reversed 24 h after multiple methamphetamine injections. Western blotting indicated that saline- and methamphetamine-affected dopamine transporters co-migrated on sodium dodecyl sulfate (SDS) gels at approximately 80 kDa, and that acute, methamphetamine-induced decreases in [3H]dopamine uptake were not due to loss of dopamine transporter protein. These findings demonstrate heretofore-uncharacterized features of the acute effect of methamphetamine on dopamine transporters.

Differential effects of psychostimulants and related agents on dopaminergic and serotonergic transporter function.

High-dose administrations of amphetamine, methamphetamine, cathinone, methcathinone or methylenedioxymethamphetamine rapidly decrease dopamine and serotonin transporter function in vivo, as assessed in striatal synaptosomes obtained from drug-treated rats. In contrast, high-dose injections of fenfluramine, cocaine or methylphenidate had little or no effect on the activity of these transporters. Interestingly, the capacity of these agents to directly alter dopamine and serotonin uptake, as assessed in vitro by direct application to rat striatal synaptosomes, did not predict their potential to modulate transporter activity following in vivo administration. These findings demonstrate heretofore-unreported differences in the effects of these agents on monoamine transporter function, and a distinction between drug effects after direct application in vitro vs. administration in vivo.

Cell-based screen for identification of inhibitors of tubulin polymerization.

This assay is based on morphological changes of rat glioma cells treated with db-cAMP. The db-cAMP treatment induces a tubulin-dependent change causing the cells to acquire a spherical shape. Pretreatment with tubulin inhibitors brings about the disintegration of tubulin polymer and/or prevents its polymerization. Cells with inhibited tubulin fail to respond to db-cAMP treatment. Cells treated with inhibitors of tubulin polymerization are then separated from the spherical cells by aspiration. A semiautomated scanning procedure evaluates the final culture density and yields graphical data.

Age-dependent differential responses of monoaminergic systems to high doses of methamphetamine.

Abuse of methamphetamine (METH) by adolescents is a major public health issue in the U.S.A. Because of the neurotoxic potential of METH, we examined the response of CNS monoaminergic systems in young (adolescent) animals [postnatal day (PND) 40] to high-dose treatments (10 mg/kg, four injections, 2-h intervals) of this drug and contrasted these effects to those seen in older (young adult) rats (PND 90). Consistent with previous reports, we observed that PND 40 animals did not manifest the long-term (7-day) deficits in extrapyramidal dopamine (DA) parameters observed in PND 90 rats. In contrast, METH-induced rapid (1-h) reduction in the activity of striatal DA transporters occurred in both age groups. In addition, both persistent (7-day) and rapid (1-h) deficits in serotonergic systems (measured as reductions in tryptophan hydroxylase activity) were observed in PND 40 and 90 rats. Age-related differences in METH-induced hyperthermia did not appear to be a principal cause for our observations; however, age-dependent pharmacokinetics of this drug might have contributed to the differential METH monoaminergic responses by PND 40 and 90 animals.

Differences in the topoisomerase I cleavage complexes formed by camptothecin and wakayin, a DNA-intercalating marine natural product.

Wakayin is bispyrroloiminoquinone isolated from a Clavelina sp. ascidian by cytotoxicity directed fractionation. Like camptothecin, it has been found to inhibit the topoisomerase I catalyzed relaxation of supercoiled DNA. Wakayin enhanced cleavage complex formation at the same DNA sequences as camptothecin. Both compounds showed dose-related increases in cleavage complex formation, though wakayin's effect is attenuated at high concentrations. Wakayin is a string DNA binder. Wakayin also differed from camptothecin in that its cleavage complexes were much less stable than those of camptothecin in 0.5 M NaCl. Again in contrast to camptothecin, wakayin stabilized cleavage complexes poorly, if at all, at 0 degree C.