Baranczyk-Kuzma A, Raub T J, Audus K L
Department of Biochemistry, Institute of Biopharmacy, Warsaw Medical School, Poland.
J Cereb Blood Flow Metab. 1989 Jun;9(3):280-9. doi: 10.1038/jcbfm.1989.46.
The existence of lysosomes and acid hydrolase activity was demonstrated in an in vitro blood-brain barrier (BBB) model comprising primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. BMEC lysosomes were observed by the uptake of acridine orange and fluorophore-labeled acetylated low-density lipoprotein by fluorescence microscopy. Cytochemical localization of the acid hydrolase, sulfatase, and acid phosphatase (AcP) activities with light microscopy also revealed hydrolase-positive vacuoles or lysosomes that varied in number from cell to cell. BMEC monolayers were fractionated and biochemical assays of the sulfatase, AcP, and beta-galactosidase were performed. Significant activities of the acid hydrolases were found to be associated with lysosome and microsome fractions (69-77%). The majority of beta-galactosidase (approximately 48%) and total sulfatase (approximately 58%) activity was associated with the lysosome fraction of the BMECs. In contrast, approximately 52% of AcP activity was associated with the microsome fraction of the cells. The results of this study are consistent with the demonstration in vivo of acid hydrolases as potential factors in the endocytic pathway for transport of proteins through the BBB and as contributors to the BBB's enzymatic barrier function.
在由牛脑微血管内皮细胞（BMEC）单层原代培养物组成的体外血脑屏障（BBB）模型中，证实了溶酶体的存在和酸性水解酶活性。通过荧光显微镜观察吖啶橙和荧光团标记的乙酰化低密度脂蛋白的摄取来观察BMEC溶酶体。用光学显微镜对酸性水解酶、硫酸酯酶和酸性磷酸酶（AcP）活性进行细胞化学定位，也揭示了水解酶阳性的液泡或溶酶体，其数量因细胞而异。对BMEC单层进行分级分离，并对硫酸酯酶、AcP和β-半乳糖苷酶进行生化分析。发现酸性水解酶的显著活性与溶酶体和微粒体部分相关（69-77%）。大部分β-半乳糖苷酶（约48%）和总硫酸酯酶（约58%）活性与BMECs的溶酶体部分相关。相比之下，约52%的AcP活性与细胞的微粒体部分相关。本研究结果与体内酸性水解酶作为蛋白质通过血脑屏障转运的内吞途径中的潜在因素以及血脑屏障酶屏障功能的贡献者的证明一致。
