有機陽離子轉運蛋白OCT2/PMAT與N-methyl-(R)SAL/1-benzyl-TIQ作用關係以及老化對腦部OCT2/PMAT表現影響之研究

Abstract

N-methyl-(R)salsolinol (N-methyl-(R)SAL) and 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-benzyl-TIQ) are endogeneous neurotoxins that can induce parkinsonism in animals. The concentrations of N-methyl-(R)SAL and 1-benzyl-TIQ in CSF are 2-3 times higher in PD patients than in control subjects. The present study was to investigate whether N-methyl-(R)SAL and 1-benzyl-TIQ can be transported by human OCT2 (hOCT2) and human PMAT (hPMAT). Also, the effects of aging on the expression of these transporters were examined. MDCKII-hOCT2 and MDCKII-hPMAT cell lines were established and the kinetic properties of N-methyl-(R)SAL and 1-benzyl-TIQ were measured. The expression of OCT2 and PMAT was measured in terms of aging and LPS treatment. Accordingly, quantitative polymerase chain reaction (qPCR) and Western blotting/immunofluorescence were used to measure mRNA and protein expression of OCT2 and PMAT, respectively. Also, the concentrations of cytokines and chemokine in the serum of aging mice were analyzed by cytometric bead array. The results showed that both 1-benzyl-TIQ and N-methyl-(R)SAL competitively inhibited MPP+ uptake in MDCKII-hOCT2 and MDCKII-hPMAT cells. 1-benzyl-TIQ was a substrate of hOCT2 (Km and Vm values of 13.5±3.7 μM and 3088.3±391.0 pmole/mg protein/min) and hPMAT (Km and Vm values of 184.0±49.9 μM and 8544.8±802.3 pmole/mg protein/min). Although N-methyl-(R)SAL can be transported by hOCT2 (Km and Vm values of 185.0±265.6 μM and 7857.9±5139.4 pmole/mg protein/min), N-methyl-(R)SAL was not a substrate of hPMAT. The values of transport efficiency (Vm/Km) suggested that 1-benzyl-TIQ was mainly transported by hOCT2 at low concentrations. OCT2 and PMAT mRNA levels were decreased in the brain of aging mice and their protein levels were also decreased in brain capillaries of aging mice. The concentrations of serum cytokines and chemokine were increased with aging indicating the association between aging and inflammation. However, OCT2 and PMAT proteins were increased in brain capillaries in mice after 24-hr LPS treatment. In conclusion, OCT2 is important for the transport of N-methyl-(R)SAL and 1-benzyl-TIQ. The down-regulation of OCT2 in brain capillaries of aging mice may result in the decrease of clearance in these neurotoxins.