Rapid loss of blood-brain barrier P-glycoprotein activity through transporter internalization demonstrated using a novel in situ proteolysis protection assay
Rapid loss of blood-brain barrier P-glycoprotein activity through transporter internalization demonstrated using a novel in situ proteolysis protection assay
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DOI：
10.1038/jcbfm.2010.117
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发表时间：
2010-09-01
影响因子：
6.3
通讯作者：
Miller, David S.
中科院分区：
文献类型：
--
作者：
Hawkins, Brian T.;Rigor, Robert R.;Miller, David S.
通过Src作用的血管内皮生长因子（VEGF）和通过蛋白激酶C（PKC）β 1作用的肿瘤坏死因子-a迅速降低血脑屏障（BBB）P-糖蛋白活性。为了探索潜在的机制，我们开发了一种基于免疫印迹的体内蛋白酶K（PK）保护试验，以评估血脑屏障管腔膜P-糖蛋白含量的变化。将PK输注到脑血管系统中选择性地切割管腔膜P-糖蛋白，使细胞内蛋白质保持完整。脑室内注射VEGF部分保护P-糖蛋白免受蛋白水解裂解，与转运蛋白内化一致。PKC β 1的激活不能保护P-糖蛋白。因此，VEGF和PKC β 1通过不同的机制降低P-糖蛋白活性。Journal of Cerebral Blood Flow & Metabolism（2010）30，1593-1597; doi：10.1038/jcbfm.2010.117; 2010年7月14日在线发表
Blood-brain barrier (BBB) P-glycoprotein activity is rapidly reduced by vascular endothelial growth factor (VEGF) acting via Src and by tumor necrosis factor-a acting via protein kinase C (PKC)beta 1. To probe underlying mechanism(s), we developed an in vivo, immunoblot-based proteinase K (PK) protection assay to assess the changes in the P-glycoprotein content of the BBB's luminal membrane. Infusion of PK into the brain vasculature selectively cleaved luminal membrane P-glycoprotein, leaving intracellular proteins intact. Intracerebroventricular injection of VEGF partially protected P-glycoprotein from proteolytic cleavage, consistent with transporter internalization. Activation of PKC beta 1 did not protect P-glycoprotein. Thus, VEGF and PKC beta 1 reduce P-glycoprotein activity by distinct mechanisms. Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1593-1597; doi:10.1038/jcbfm.2010.117; published online 14 July 2010