The Interaction Between Dopamine And Dopamine Receptor Agonist Based On Organic Cation Transporter 2/3

Parkinson's disease(PD)has been the second largest neurodegenerative disease around the world,seriously threatening health and life of the elderly.PD is typically characterized by progressive damage to the nigrostriatal dopaminergic neurons,with the enormous depletion of striatal dopamine(DA).Pramipexole(PPX)and ropinirole(RPR)are non-ergotine selective dopamine receptor agonists,which have been widely used in clinical anti-PD treatment.However,long-term treatment with PPX and RPR usually results in declined therapeutic effects and finally limitation of their clinical application.Nevertheless,few researchs has been reported about the mechanism of dopamine receptor agonist tolerance under long-term medication.Previous studies have shown that organic cation transporter 2/3(OCT2/3)can regulate the homeostasis of DA in brain when dopamine transporters were greatly depleted under the PD state.Meanwhile,PPX has been proven a substrate of OCT2 and OCT3.Therefore,this study focused on the possible interaction between DA and PPX/RPR based on OCT2/3,explored whether OCT2 and OCT3 were potential target of the treatment of PPX/RPR,evaluated the changes of OCT2/3 expression and function under PD status,and finally discussed the role of OCT2/3 in the treatment as well as declined therapeutic effects of PPX/RPR when long-term used.Firstly,two LC-MS/MS quantitative analysis methods of DA in cell samples and brain tissue homogenate samples were established respectively.Surrogate analyte method was employed with a view of the inteference of endogenous DA when analyzing brain homogenate samples.In brief,d4-DA was used as the surrogate analyte and d2-DA as the internal standard.Method validation revealed that the very two methods showed high sensitivity and strong selectivity with acceptable accuracy and precision.The coefficient of variation of matrix effect and recovery was less than 15%.Furthermore,both methods produced desired reproducibility and stability.In addition,the analysis time of the two methods is only 3.5 minutes,promising a high-throughput sample analysis.The successful method development and validation laid a foundation for the sensitive,accurate and rapid quantitative analysis of the subsequent experimental samples.Time-and concentration-dependence of DA,PPX,RPR uptake mediated via MDCKOCT2 and MDCK-OCT3 cells were conducted to confirm whether DA,PPX,and RPR are candidate substrate of OCT2/3.Results showed that the uptake of three compouds by transfected cells was time and concentration saturable with a good fit to Michaelis equation,and significantly higher than that of cells transfected with empty vector,except that the RPR uptake by MDCK-OCT3 cells shared no statistical difference with mock group.Subsequently inhibition study showed that various OCT2/3 inhibitors were capable of significantly inhibiting the DA,PPX,and RPR uptake by transfected cells,which illustrated that both DA and PPX are substrates of OCT2 as well as OCT3,and RPR is a substrate of OCT2 but not OCT3.Then the interaction between DA and PPX/RPR based on OCT2 and OCT3 were investigated.The results showed that PPX and RPR could inhibit DA uptake by OCT2/3 with a double-site inhibition feature on OCT2;DA in return showed a certain inhibitory effect on PPX uptake by OCT2/3 and RPR uptake by OCT2.Above results suggest that OCT2/3 may be potential targets for PPX/RPR.The anti-PD pharmacological activity of PPX and RPR can be achieved by inhibiting DA reuptake via OCT2/3 followed by an increase of synaptic cleft DA level.In return,as the striatal DA decreased under PD state,PPX and RPR uptake by OCT2/3 would be enhanced for the abcence of DA inhibition,leading to faster clearance of PPX and RPR,which may be one of the reasons for the declined therapeutic effects during the long-term medication of PPX and RPR.Given the important role of OCT2/3 in PD,a PD cellular model employed BV2 cells was constrcucted to evaluated the changes in Oct2 and Oct3 under PD status from three aspects: gene level,protein expression and transport function.The results of RT-q PCR and western blot tests showed that the expression of Oct2 and Oct3 genes and proteins in BV2 cells were significantly enhanced.Subsequent inhibition experiments showed that DA and PPX/RPR interact on BV2 cells,which was consistent with previous experimental results.Moreover,the uptake of metformin(MTF,a classical probe of Octs),DA,PPX and RPR by model group BV2 cells were obviously higher than control group cells,and identical concentration of OCT2/3 inhibitors showed strengthened inhibitory effects on model group cells.The molecular pharmacokinetics of BV2 cells mediated uptake showed that in addition to the enhancement of expression,the function of Oct2/3 on BV2 cells was also up-regulated.These results suggest that the expression and function of OCT2/3 might be up-regulated under PD state,which will accelerate the clearance of DA,PPX and RPR from synaptic cleft,and finally lead to the declined effects of PPX and RPR.Whereafter,PD mice model was constructed and validated based on behavior,histology and striatal DA level.Then the gene and protein expression of OCT2/3 in the striatum of model group and control group were evaluated by RT-q PCR and western blot tests respectively.Finally,the synaptosomes were extracted and applied to the uptake experiments of MTF,d4-DA,PPX and RPR.Results of PD model validation showed that the mice in model group had PD like motor symptoms,while dopaminergic neurons in substantia nigra and striatum were significantly reduced,and the DA level in striatum was also reduced to 12% of that in the control group.RT-q PCR and western blot tests showed that the expression of Oct2 and Oct3 gene and protein in the striatum of the model group mice was significantly higher than that of the control group.The subsequent uptake and inhibition experiments showed that the uptake of MTF,d4-DA,PPX and RPR by synaptosomes in model group was significantly higher,and the uptake was more easily inhibited by OCT2/3 inhibitors.In addition,there were also obvious interactions between d4-DA and PPX/RPR on synaptosomes.The above studies further confirmed the experimental results of BV2 cells at animal level,and showed a strong proof for the mechanism of anti-PD as well as declined therapeutic effect of PPX and RPR mediated by OCT2/3.In order to explore the mechanism of function changes of OCT2/3 under PD status based on protein-protein interaction(PPI),the protein tertiary structure model of OCT2 and OCT3 was prediceted using bioinformatics technology and comprehensively optimized.Then prediction of the interaction between OCT2/3 and PD closely related proteins was conducted based on the information of sequence,domain and interaction network.Three proteins,DJ-1,GSTO-1,ALDH1A1,which may interact with OCT3 and two proteins,BST1 and DRP-1,which may interact with OCT3,were screened out.Then the PPI active sites of these seven proteins were predicted,and the corresponding protein complexes were generated by molecular docking according to the predicted sites.Finally,the most reasonable protein complex conformation was obtained by binding energy and ASA screening.The above study of OCT2/3 PPI based on bioinformatics could be the guidence for further screening and research of OCT2/3 related PPI by using experimental methods,and lays a foundation for studying the mechanism of OCT2/3 function change under PD state based on PPI.