Hartz Anika M S, Bauer Björn, Fricker Gert, Miller David S
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Mol Pharmacol. 2004 Sep;66(3):387-94. doi: 10.1124/mol.104.001503.
The ATP-driven xenobiotic transporter P-glycoprotein is a critical element of the blood-brain barrier. To study regulation of P-glycoprotein function, we measured specific transport [(3'-oxo-4-butenyl-4-methyl-threonine(1), (valine(2)) cyclosporin (PSC833)-sensitive] of the fluorescent cyclosporin A derivative [N-epsilon(4-nitrobenzofurazan-7-yl)-D-Lys(8)]-cyclosporin A (NBDL-CSA) into the lumens of isolated rat brain capillaries using confocal microscopy and quantitative image analysis. Luminal NBDL-CSA accumulation was rapidly and reversibly reduced in a concentration-dependent manner by 0.1 to 100 nM endothelin-1 (ET-1). In this concentration range, ET-1 did not affect junctional permeability. The ET(B) receptor agonist sarafotoxin 6c also reduced transport. An ET(B) receptor antagonist blocked effects of ET-1 and sarafotoxin 6c; an ET(A) receptor antagonist was without effect. Consistent with this, immunostaining and Western blotting showed expression of the ET(B) receptor in brain capillary membranes. NBDL-CSA transport was also reduced by sodium nitroprusside, a NO donor, and by phorbol ester, a protein kinase C (PKC) activator. Inhibition of NO synthase (NOS) or PKC abolished the ET-1 effects. Thus, ET-1, acting through an ET(B) receptor, NOS, and PKC rapidly and reversibly reduced transport mediated by P-glycoprotein at the blood-brain barrier.
ATP驱动的外源性物质转运体P-糖蛋白是血脑屏障的关键组成部分。为研究P-糖蛋白功能的调节机制，我们使用共聚焦显微镜和定量图像分析技术，测量了荧光环孢素A衍生物[N-ε(4-硝基苯并呋喃唑-7-基)-D-赖氨酸(8)]-环孢素A（NBDL-CSA）向分离的大鼠脑毛细血管管腔中的特异性转运[（3'-氧代-4-丁烯基-4-甲基苏氨酸(1)，（缬氨酸(2)）环孢素（PSC833）敏感]。内皮素-1（ET-1）以浓度依赖的方式迅速且可逆地降低了管腔内NBDL-CSA的积累，浓度范围为0.1至100 nM。在此浓度范围内，ET-1不影响连接通透性。ET(B)受体激动剂沙拉毒素6c也降低了转运。ET(B)受体拮抗剂可阻断ET-1和沙拉毒素6c的作用；ET(A)受体拮抗剂则无此作用。与此一致的是，免疫染色和蛋白质印迹显示ET(B)受体在脑毛细血管膜中有表达。硝普钠（一种NO供体）和佛波酯（一种蛋白激酶C（PKC）激活剂）也降低了NBDL-CSA的转运。抑制一氧化氮合酶（NOS）或PKC可消除ET-1的作用。因此，ET-1通过ET(B)受体、NOS和PKC作用，迅速且可逆地降低了血脑屏障处由P-糖蛋白介导的转运。
