Organic cation transporters mediate pharmacokinetic interaction between dopamine receptor agonists and dopamine

Abstract

Purpose Parkinson's disease (PD) is typically characterized by progressive damage to the nigrostriatal dopaminergic neurons with the enormous depletion of striatal dopamine (DA). DA receptor agonists such as pramipexole (PPX) and ropinirole (RPR) have been widely used in clinic. However, long-term treatment with DA receptor agonists often results in declined therapeutic effect. Recent research has shown that uptake-2 transporters including organic cation transporters (OCT) 2/3 play an important role in the regulation of DA homeostasis in brain. The aim of the present study was to investigate the underlying mechanism by which DA receptor agonists modulate DA homeostasis. Methods Transfected cells were incubated with HBSS containing serial concentration of PPX/RPR/DA with or without OCT2/3 inhibitors. LC-MS/MS was used to quantify the level of substrates in cell lysates. Then, male C57BL/6J mice received MPTP subcutaneously to construct PD model. Synaptosomes were extracted by sucrose density gradient centrifugation, incubated with uptake buffer containing predefined substrate and inhibitor and precipitated after 5 min. Lysed followed by LC-MS/MS quantification. Striatal Oct2/3 gene and protein levels were measured by RT-qPCR and western blotting, respectively. Finally, the tertiary structure models of OCT2/3 were predicted and optimized using bioinformatics technology. The molecular docking between OCT2/3 and substrates of interest were predicted by ClusPro servers. Results Both MDCK-OCT2 and MDCK-OCT3 cells demonstrated significantly higher cellular uptake of DA/PPX compared with MDCK mock cells. Also, RPR uptake was increased when OCT2 was overexpressed. A variety of classical OCT2/3 inhibitors decreased cellular uptake of DA/PPX/RPR. Furthermore, PPX/RPR could inhibit OCT2/3 mediated DA uptake following distinct kinetic processes. On the other hand, OCT2-mediated PPX/RPR uptake could also be restrained by DA. The interaction between DA and PPX/RPR was further confirmed at the synatosomes level. Besides, OCT2/3 expression and function were obviously elevated under PD status and thus the transport efficiency was increased. The molecular docking results revealed the OCT2/3 binding pockets as well as affinity difference between DA receptor agonists and DA. The affinity results were in good agreement with the findings of uptake assay. Conclusion This study not only offers a novel perspective on anti-PD mechanism of PPX and RPR as organic cation transporter inhibitor, but also demonstrated that up-regulated OCT2 and OCT3 might be associated with the declined therapeutic effect of PPX and RPR.