Calhau C, Martel F, Soares-da-Silva P, Hipólito-Reis C, Azevedo I
Departamento de Bioquímica, Faculdade de Medicina. 4200-319 Porto, Portugal.
Naunyn Schmiedebergs Arch Pharmacol. 2002 May;365(5):349-56. doi: 10.1007/s00210-002-0542-1. Epub 2002 Mar 26.
The aim of this study was to investigate the role of phosphorylation/dephosphorylation mechanisms at the blood-brain barrier (BBB) in the uptake of organic cations. The experiments were performed using RBE4 cells, an immortalized, rat brain microvessel endothelial cell line, an in vitro model of the BBB. The modulation of the uptake of 1-methyl-4-phenylpyridinium (MPP(+)), a model organic cation, at the apical membrane of RBE4 cells was studied. Agents that stimulate protein kinase A, but not protein kinase C, produced a moderate inhibition (approximately 18% reduction) of uptake of [(3)H]MPP(+) by RBE4 cells. Okadaic acid, an inhibitor of protein serine/threonine phosphatase, did not affect uptake of (3)H-MPP(+), but the alkaline phosphatase (ALP) inhibitor levamisole markedly reduced (3)H-MPP(+) uptake. The activity of membrane-bound ALP expressed on the apical surface of RBE4 cells was studied at pH 7.4 using p-nitrophenylphosphate as substrate. Kaempferol, progesterone, 3-isobutyl-1-methylxanthine, all- trans-retinoic acid and iron stimulated ecto-ALP activity and uptake of [(3)H]MPP(+) in RBE4. Orthovanadate (a protein tyrosine phosphatase inhibitor) markedly inhibited both ecto-ALP activity and uptake of [(3)H]MPP(+) by RBE4 cells. In conclusion, these results suggest that apical transporter(s) of MPP(+) in RBE4 cells may be under the control of phosphorylation/dephosphorylation mechanisms, being active in the dephosphorylated state. A physiological role for ALP in the modulation of organic cation transport in the BBB is suggested.
本研究的目的是调查血脑屏障（BBB）处的磷酸化/去磷酸化机制在有机阳离子摄取中的作用。实验使用RBE4细胞进行，RBE4细胞是一种永生化的大鼠脑微血管内皮细胞系，是血脑屏障的体外模型。研究了RBE4细胞顶膜上1-甲基-4-苯基吡啶鎓（MPP(+)）（一种典型的有机阳离子）摄取的调节情况。刺激蛋白激酶A而非蛋白激酶C的试剂对RBE4细胞摄取[(3)H]MPP(+)产生了中度抑制（约18%的降低）。蛋白丝氨酸/苏氨酸磷酸酶抑制剂冈田酸不影响(3)H-MPP(+)的摄取，但碱性磷酸酶（ALP）抑制剂左旋咪唑显著降低了(3)H-MPP(+)的摄取。使用对硝基苯磷酸作为底物，在pH 7.4条件下研究了RBE4细胞顶表面表达的膜结合ALP的活性。山奈酚、孕酮、3-异丁基-1-甲基黄嘌呤、全反式维甲酸和铁刺激了RBE4细胞的胞外ALP活性及[(3)H]MPP(+)的摄取。原钒酸盐（一种蛋白酪氨酸磷酸酶抑制剂）显著抑制了RBE4细胞的胞外ALP活性及[(3)H]MPP(+)的摄取。总之，这些结果表明RBE4细胞中MPP(+)的顶端转运体可能受磷酸化/去磷酸化机制的控制，在去磷酸化状态下具有活性。提示了ALP在血脑屏障中有机阳离子转运调节中的生理作用。
