ABSTRACT
Ring size reduction of the central piperidine ring of lobelane yielded pyrrolidine analogues that showed marked inconsistencies in their ability to bind to the dihydrotetrabenazine (DTBZ) binding site on the vesicular monoamine transporter-2 (VMAT2) and their ability to inhibit VMAT2 function. The structure-activity relationships indicate that structural modification within the pyrrolidine series resulted in analogues that interact with two different sites, i.e., the DTBZ binding site and an alternative site on VMAT2 to inhibit transporter function.
Subject(s)
Lobeline/analogs & derivatives , Pyrrolidines/chemistry , Pyrrolidines/metabolism , Tetrabenazine/analogs & derivatives , Vesicular Monoamine Transport Proteins/chemistry , Vesicular Monoamine Transport Proteins/metabolism , Binding Sites , Lobeline/chemistry , Protein Binding , Pyrrolidines/chemical synthesis , Pyrrolidines/pharmacology , Structure-Activity Relationship , Tetrabenazine/metabolism , Vesicular Monoamine Transport Proteins/antagonists & inhibitorsABSTRACT
The molecular mechanism of 1-methyl-4-phenylpyridinium (MPP+), a Parkinsonism-inducing neurotoxin, has been studied in PC12 cells. The cells treated with MPP+ (100 microM) induced a rapid increase in phosphorylation of tyrosine residues of several proteins, including synaptophysin, a major 38 kDa synaptic vesicle protein implicated in exocytosis. An accelerated release of dopamine by MPP+ correlated with phosphorylation of synaptophysin. Exposing the cells to MPP+ triggered reactive oxygen species (ROS) generation within 60 min of treatment and the said effect was blocked by mazindol, a dopamine uptake blocker. In addition, pretreatment with 50-100 microM of selegiline, a selective MAO-B inhibitor, significantly suppressed MPP+-mediated ROS generation. These effects of MPP+ result in the generation of ROS, which may be involved in neuronal degeneration seen in Parkinson's disease.
Subject(s)
1-Methyl-4-phenylpyridinium/pharmacology , Dopamine/metabolism , Adrenal Medulla/drug effects , Adrenal Medulla/metabolism , Animals , Calcium/metabolism , Dopamine Uptake Inhibitors/pharmacology , Exocytosis , Mazindol/pharmacology , Monoamine Oxidase Inhibitors/pharmacology , PC12 Cells , Phosphorylation , Rats , Reactive Oxygen Species , Selegiline/pharmacology , Synaptophysin/metabolism , Tyrosine/metabolismABSTRACT
The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP(+)) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP(+)-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP(+) produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP(+). The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B(max)) with an increase in the dissociation equilibrium constant (K(d)) after MPP(+) treatment. Furthermore, biotinylation assays confirmed that MPP(+) reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP(+) may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.
