Parkinson's disease (PD) is a progressive disease related to degeneration of nigrostriatal dopaminergic neurons and can be caused by genetic and environmental factors. N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can produce Parkinsonian syndrome (Langston et al. 1983) and is an Received February 28, 2010; revised manuscript received April 26, 2010; accepted April 26, 2010. Address correspondence and reprint requests to Kai-Yuan Tzen, MD, Department of Nuclear Medicine, National Taiwan University Hospital, No.7 Chung Shan South Road, Taipei 100, Taiwan. E-mail: tzenky@ntuh.gov.tw or Chun-Jung Lin, School of Pharmacy, College of Medicine, National Taiwan University,1 Jen-Ai Road, Section 1, Taipei, Taiwan 100. E-mail: clementumich@ntu.edu.tw Abbreviations used: 3-MT, 3-methoxytyramine; ARBECs, adult rat brain endothelial cells; AUC, area under the time-concentration curve; BB, blood-brain barrier; BMECs, brain microvessel endothelial cells; BSA, bovine serum albumin; DC, detergent compatible; DOPAC, 3,4-dihydroxyphenylacetic acid; ECF, extracellular fluid; FDOPA, 6-[ AbstractThe cellular localization of organic cation transporter (OCT) 1 and OCT2 in isolated brain microvessel endothelial cells from humans, rats, and mice and in cultured adult rat brain endothelial cells was examined by confocal microscopy and in isolated luminal and abluminal membrane fractions by Western blot analysis. Cellular uptake of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was measured with or without OCT1/OCT2 silencing. The interaction between MPTP and amantadine was studied by in vitro kinetic analysis and in vivo brain microdialysis. MPTP-induced dopaminergic toxicity was examined by measuring dopamine levels in the brain striatum and by positron emission tomography scanning. The results showed that both OCT1 and OCT2 were mainly expressed on the luminal side of brain microvessel endothelial cells and adult rat brain endothelial cells. Cellular uptake of MPTP was significantly (p < 0.05) decreased by about 53%, 60%, or 91% following silencing of OCT1, OCT2, or both, respectively. Amantadine competitively inhibited MPTP uptake in vitro and significantly (p < 0.05) reduced the area under the time-concentration curve for MPTP and MPP + in the brain extracellular fluid in rats and mice by 65-70% and 35-85%, respectively. MPTP-induced dopaminergic toxicity in mice was ameliorated by amantadine without stimulating dopamine turnover. In conclusion, OCT1 and OCT2 are important for MPTP transfer across the blood-brain barrier and amantadine reduces the blood-brain barrier transfer of MPTP and MPTP-induced dopaminergic toxicity in rodents.

The intestinal facilitated glucose transporter, GLUT2, is a high-turnover transport system and is important to handle the large transepithelial substance flux. Since intestinal GLUT2 is normally located at the basolateral side, glucose uptake in the presence of flavonoids was measured using basolateral membrane vesicles (BLMV) isolated from the rat jejunum to investigate the interaction between flavonoids and intestinal facilitated glucose transporters. In addition, basolateral uptake of flavonoids was studied in Caco-2 cells. As a result, in the BLMV study most flavonoids (glycosides or aglycones) at 0.1 mM inhibited glucose uptake in BLMV; epicatechin gallate (ECG) showed the highest inhibitory activity (about 33%), followed by quercetin 3-O-glucoside (Q3G), fisetin and gossypin (about 25-28% inhibition). The dose-response study showed that the IC50 values for ECG and Q3G on glucose uptake in BLMV were 294+/-89 microM and 357+/-52 microM, respectively. Kinetic analyses showed that ECG and Q3G competitively inhibited glucose uptake in BLMV with inhibition constants (Ki) of 332+/-42 microM and 404+/-45 microM, respectively. In Caco-2 cells, basolateral uptake of Q3G was significantly inhibited by phloretin, a GLUT2 inhibitor (0.40+/-0.05 vs. 0.24+/-0.03 nmole/mg protein without aand with phloretin, respectively). On the other hand, phloretin did not show inhibitory activity on basolateral uptake of ECG in Caco-2 cells (1.26+/-0.05 vs. 1.22+/-0.07 nmole/mg protein without and with phloretin, respectively). The data showed that the intestinal facilitated glucose transporter recognizes a variety of flavonoids with or without conjugation. In addition, GLUT2 can be responsible for the transport of Q3G across the intestinal basolateral membrane.

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