Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
J Cereb Blood Flow Metab. 2010 Jul;30(7):1373-83. doi: 10.1038/jcbfm.2010.21. Epub 2010 Mar 3.
P-glycoprotein is an ATP (adenosine triphosphate)-driven drug efflux transporter that is highly expressed at the blood-brain barrier (BBB) and is a major obstacle to the pharmacotherapy of central nervous system diseases, including brain tumors, neuro-AIDS, and epilepsy. Previous studies have shown that P-glycoprotein transport activity in rat brain capillaries is rapidly reduced by the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha) acting through protein kinase C (PKC)-dependent signaling. In this study, we used isolated rat brain capillaries to show that the TNF-alpha-induced reduction of P-glycoprotein activity was prevented by a PKCbeta(I/II) inhibitor, LY333531, and mimicked by a PKCbeta(I/II) activator, 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA). Western blotting of brain capillary extracts with phospho-specific antibodies showed that dPPA activated PKCbeta(I), but not PKCbeta(II). Moreover, in intact rats, intracarotid infusion of dPPA potently increased brain accumulation of the P-glycoprotein substrate, [(3)H]-verapamil without compromising tight junction integrity. Thus, PKCbeta(I) activation selectively reduced P-glycoprotein activity both in vitro and in vivo. Targeting PKCbeta(I) at the BBB may prove to be an effective strategy for enhancing the delivery of small molecule therapeutics to the brain.
P-糖蛋白是一种 ATP（三磷酸腺苷）驱动的药物外排转运蛋白，在血脑屏障（BBB）中高度表达，是中枢神经系统疾病（包括脑肿瘤、神经艾滋病和癫痫）药物治疗的主要障碍。先前的研究表明，促炎细胞因子肿瘤坏死因子-α（TNF-α）通过蛋白激酶 C（PKC）依赖性信号转导作用，可迅速降低大鼠脑毛细血管中的 P-糖蛋白转运活性。在这项研究中，我们使用分离的大鼠脑毛细血管表明，PKCβ（I/II）抑制剂 LY333531 可预防 TNF-α诱导的 P-糖蛋白活性降低，PKCβ（I/II）激活剂 12-脱氧佛波醇-13-苯乙酸-20-乙酸酯（dPPA）可模拟该作用。用磷酸化特异性抗体对脑毛细血管提取物进行的 Western blot 分析表明，dPPA 激活了 PKCβ（I），但不激活 PKCβ（II）。此外，在完整的大鼠中，dPPA 经颈动脉内输注可强力增加 P-糖蛋白底物[3H]-维拉帕米在脑内的蓄积，而不会损害紧密连接的完整性。因此，PKCβ（I）的激活无论是在体外还是在体内都选择性地降低了 P-糖蛋白的活性。在 BBB 靶向 PKCβ（I）可能被证明是一种有效的策略，可增强小分子治疗药物向大脑的递送。
