Thompson S E, Cavitt J, Audus K L
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Kansas, Lawrence 66045-2504.
J Cardiovasc Pharmacol. 1994 Nov;24(5):818-25. doi: 10.1097/00005344-199424050-00018.
Leucine enkephalin (YGGFL) effects on markers for transcellular and paracellular permeation across the blood-brain barrier (BBB) were investigated in vitro with bovine brain microvessel endothelial cell (BMEC) monolayers in primary culture. Intact YGGFL, but not metabolites of YGGFL, stimulated BMEC uptake of lucifer yellow (LY), a marker for fluid-phase endocytosis, in a concentration-dependent manner. However, D-[Ala2]-leucine enkephalin (YAGFL), a YGGFL analogue that altered BMEC monolayer permeability, had no effect on LY uptake. In part, these results suggested that YGGFL's effects on fluid-phase uptake might not relate directly to enhanced BMEC transcellular permeability in the presence of the peptide. The measurement of the fluorescence anisotropy of membrane-bound diphenyl-hexatriene probes did not show substantial peptide-induced changes in membrane lipid packing order (i.e., membrane fluidity) and indicated a limited role for membrane perturbations in YGGFL-induced changes in BMEC monolayer permeability. Conversely, the apparent permeability coefficients showed size-dependent YGGL-induced alterations for passage of membrane-impermeant substances across BMEC monolayers. The apparent permeability coefficients of low-molecular-weight (low-mol-wt) molecules (mannitol, sucrose, and fluorescein) were increased on exposure to YGGFL. The apparent permeability coefficients for high-mol-wt molecules, FITC dextran conjugates (4, 20, and 71.6 Kd), were not affected by exposure to YGGFL. Transmission electron micrographs of lanthanum (Stoke's radius, 10 A) exclusion from BMEC intercellular junctions supported these observations. Collectively, results from this study suggest that YGGFL enhanced BMEC permeability either by altering paracellular openings or through formation of a small pore in the monolayers to allow preferential penetration of low-mol-wt or small molecular size (< 10 A) substances.
在体外，使用原代培养的牛脑微血管内皮细胞（BMEC）单层，研究了亮氨酸脑啡肽（YGGFL）对跨血脑屏障（BBB）的跨细胞和细胞旁渗透标志物的影响。完整的YGGFL，而非YGGFL的代谢产物，以浓度依赖的方式刺激BMEC摄取荧光素黄（LY），LY是液相内吞作用的标志物。然而，改变BMEC单层通透性的YGGFL类似物D-[Ala2]-亮氨酸脑啡肽（YAGFL）对LY摄取没有影响。这些结果部分表明，YGGFL对液相摄取的影响可能与肽存在时BMEC跨细胞通透性增强没有直接关系。膜结合的二苯基己三烯探针的荧光各向异性测量未显示肽诱导的膜脂堆积顺序（即膜流动性）有实质性变化，表明膜扰动在YGGFL诱导的BMEC单层通透性变化中作用有限。相反，表观渗透系数显示YGGFL诱导的膜不通透性物质跨BMEC单层通过的变化与大小有关。低分子量（低mol-wt）分子（甘露醇、蔗糖和荧光素）的表观渗透系数在暴露于YGGFL时增加。高分子量分子FITC葡聚糖缀合物（4、20和71.6 Kd）的表观渗透系数不受暴露于YGGFL的影响。镧（斯托克斯半径，10 Å）从BMEC细胞间连接排除的透射电子显微镜图像支持了这些观察结果。这项研究的结果共同表明，YGGFL通过改变细胞旁开口或在单层中形成小孔以允许低mol-wt或小分子大小（<10 Å）物质优先渗透来增强BMEC通透性。
