Interactions of 1-methyl-4-phenylpyridinium and other compounds with P-glycoprotein: relevance to toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

The vesicular monoamine transporter 2 (VMAT2) has sequence homology with bacterial multidrug transporters which in turn share homology with mammalian P-glycoprotein (P-GP). Both VMAT2 and P-GP can detoxify cells. 1-Methyl-4-phenylpyridinium (MPP(+)), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a substrate for VMAT2 that has several structural features in common with P-GP substrates and inhibitors. The present studies investigated whether P-GP is responsible for the elimination of MPP(+) from the brain. Additionally, VMAT2 and P-GP are inhibited by many of the same compounds. Thus we also investigated whether VMAT2 inhibitors could block P-GP in vitro and vice versa whether P-GP inhibitors could block VMAT2 mediated transport of [3H]-DA into synaptic vesicles. In mice treated with MPTP and a P-GP inhibitor (quinidine, trans-flupentixol or cyclosporine A), the elimination of MPP(+) from the striatum was significantly delayed. However, in experiments using various cell lines expressing either mouse or human P-GP, MPP(+) did not reverse the P-GP mediated resistance to vincristine, suggesting that MPP(+) is a poor substrate for P-GP. Additional experiments were performed using mdr1a/b double knockout mice which lack functional P-GP encoded by these two genes. Data from mdr1a/b knockout mice treated with MPTP also suggest that MPP(+) is not extruded from the brain by P-GP. In other studies, we demonstrated that the VMAT2 inhibitors tetrabenazine and Ro 4-1284 inhibit P-GP and that the P-GP inhibitors trans-flupentixol and quinidine inhibit VMAT2. Thus, several new drugs can be added to the list of compounds that are able to inhibit both VMAT2 and P-GP, providing further evidence of the similarity between these two transporters.

Neuroprotection by caffeine and A(2A) adenosine receptor inactivation in a model of Parkinson's disease.

Recent epidemiological studies have established an association between the common consumption of coffee or other caffeinated beverages and a reduced risk of developing Parkinson's disease (PD). To explore the possibility that caffeine helps prevent the dopaminergic deficits characteristic of PD, we investigated the effects of caffeine and the adenosine receptor subtypes through which it may act in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin model of PD. Caffeine, at doses comparable to those of typical human exposure, attenuated MPTP-induced loss of striatal dopamine and dopamine transporter binding sites. The effects of caffeine were mimicked by several A(2A) antagonists (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261), 3,7-dimethyl-1-propargylxanthine, and (E)-1,3-diethyl-8 (KW-6002)-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione) (KW-6002) and by genetic inactivation of the A(2A) receptor, but not by A(1) receptor blockade with 8-cyclopentyl-1,3-dipropylxanthine, suggesting that caffeine attenuates MPTP toxicity by A(2A) receptor blockade. These data establish a potential neural basis for the inverse association of caffeine with the development of PD, and they enhance the potential of A(2A) antagonists as a novel treatment for this neurodegenerative disease.

Analysis of VMAT2 binding after methamphetamine or MPTP treatment: disparity between homogenates and vesicle preparations.

[3H]Dihydrotetrabenazine ([3H]DTBZ), a specific ligand for the vesicular monoamine transporter (VMAT2), has been used to characterize the integrity of monoaminergic nerve terminals in experimental animals and humans. The purpose of the present studies was to compare the loss of VMAT2 binding with the loss of other neurochemical markers of the dopamine (DA) nerve terminals in mice treated with neurotoxic doses of methamphetamine (METH) or MPTP. Profound decreases (> or =70%) in DA content, tyrosine hydroxylase activity, and PH]carbomethoxy-3-(4-fluorophenyl)tropane binding to the DA transporter were observed in striatal homogenates at both 1 and 6 days after exposure to the neurotoxins. It is surprising that no significant loss of [3H]DTBZ binding in the homogenates was observed at 1 day after exposure, although a significant loss (-50%) was apparent 6 days later. However, in isolated vesicle preparations, [3H]DTBZ binding and active [3H]DA uptake were markedly reduced (>70%) at 1 day. These observations indicate that vesicle function is compromised at an early time point after exposure to neurotoxic insult. Furthermore, the changes in [H]DTBZ binding in homogenates may not be a sensitive indicator of early damage to synaptic vesicles, although homogenate binding reliably identifies a loss of VMAT2 at later times.

Inhibition of brain vesicular monoamine transporter (VMAT2) enhances 1-methyl-4-phenylpyridinium neurotoxicity in vivo in rat striata.

Dopamine neurons from various animal species differ in sensitivity to the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP(+)). Compared with striatal vesicles isolated from mice, those from rats have a higher density of the brain vesicular monoamine transporter (VMAT2) and a greater ability to sequester MPP(+), suggesting a larger storage capacity for MPP(+) in rat vesicles. In the present study, we examined whether striatal VMAT2-containing vesicles might provide protection against the neurotoxic effects of MPP(+) in vivo. Dose-response curves for striatally infused MPP(+) were determined in animals pretreated with or without a VMAT2 inhibitor. Ro 4-1284 administration (10 mg/kg i.p.; VMAT2 inhibitor) produced a 5-fold leftward shift in the MPP(+) dose-response curve and a significant lowering of the EC(50) concentration for MPP(+)-induced damage. These findings provide evidence for a substantial accumulation of MPP(+) in VMAT2-containing vesicles in vivo in the rat striatum and support the hypothesis that MPP(+) sequestration in vesicles can provide protection against its toxic actions. In mice, VMAT2 inhibition did not reliably enhance toxicity produced by a striatal infusion of MPP(+) or by systemic administration of MPTP. These data suggest that vesicular sequestration of MPP(+) may be of less importance in mice than in rats as relates to protection from the toxin. The present results also reveal that although VMAT2 inhibition enhanced striatal MPP(+) toxicity in the rat, the potency of MPP(+) in the rat striatum was less than that in mouse striatum. This implies that there are other factors that either exacerbate MPP(+) toxicity in the mouse or attenuate MPP(+) toxicity in rats.

In vitro studies of striatal vesicles containing the vesicular monoamine transporter (VMAT2): rat versus mouse differences in sequestration of 1-methyl-4-phenylpyridinium.

Significant differences exist in the sensitivity of mice and rats to the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that cannot be explained by differences in exposure to or uptake of 1-methyl-4-phenylpyridinium (MPP(+)) into dopamine (DA) neurons. MPP(+) is also a substrate for the brain vesicular monoamine transporter (VMAT2), and sequestration into synaptic vesicles may be one mechanism of protection against MPP(+) toxicity. A greater sequestration of MPP(+) into vesicles of DA neurons in rats versus mice could explain the lower vulnerability of DA neurons in the rat to MPP(+) toxicity. To test this hypothesis, the kinetics of uptake for [(3)H]MPP(+) and [(3)H]DA as well as [(3)H]dihydrotetrabenazine binding to VMAT2 were compared in vesicles isolated from the striata of rats and mice. The K(m) value of [(3)H]MPP(+) transport was similar in the two species. In contrast, the maximal transport rate (V(max)) was 2-fold greater in vesicles from rats than in those from mice. Likewise, the K(m) value for [(3)H]DA transport was similar in both preparations, but the V(max) value was 2-fold greater in rat than in mouse vesicles. The B(max) value for [(3)H]dihydrotetrabenazine binding was also 2-fold greater in striatal vesicles from rats than in those from mice. Electron micrographs demonstrated that vesicles isolated from rats and mice were approximately the same size. Based on these observations, we propose that striatal vesicles from rats have more VMAT2 than vesicles from mice and that this species difference in VMAT2 density may help explain the reduced vulnerability of rat DA neurons to MPP(+) neurotoxicity.