Matsukawa Y, Yamahara H, Lee V H, Crandall E D, Kim K J
Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, USA.
Pharm Res. 1996 Sep;13(9):1331-5. doi: 10.1023/a:1016013731237.
To evaluate the transport characteristics of horseradish peroxidase (HRP, a nonspecific fluid-phase endocytosis marker) across an in vitro model of tight (> 2,000 ohm-cm2) rat alveolar epithelial cell monolayers grown on tissue culture-treated polycarbonate filters.
Unidirectional HRP fluxes were estimated from the appearance rate of HRP in the receiver fluid following instillation in the donor fluid as a function of donor [HRP] and temperature. Molecular species present in either bathing fluid were determined at the end of flux experiments using fluorescein isothiocyanate (FITC)-labeled HRP by gel permeation chromatography. Cell-associated HRP activity at the end of the transport experiment was determined, as were the rates of recycling and transcellular movement of HRP. An enzymatic assay was uses to quantify HRP activity in the bathing fluid and cells.
Unidirectional HRP fluxes were symmetric and increased linearly with up to 50 microM donor [HRP]. The apparent permeability coefficient of HRP was reduced by 3.5 times upon lowering the temperature from 37 to 4 degrees C. About 50% of the FITC-labeled species present in either receiver fluid was intact HRP. Cell-associated HRP estimated from apical HRP incubation was about 4 times greater than that from basolateral incubation. Recycling into apical fluid of cell-associated HRP following apical incubation occurred rapidly with a half-time (T1/2) of approximately 5 min, reaching a plateau at approximately 67% of the initial cell-associated HRP, while transcellular movement of HRP (into basolateral fluid) took place with a T1/2 of approximately 20 min, attaining a steady-state at approximately 13% of the initial cell-associated HRP. Basolateral recycling of HRP was also rapid (T1/2 = approximately 5 min) reaching a steady-state at approximately 35% of the initial basolaterally-bound HRP. Transcellular movement of HRP following basolateral incubation was slower (T1/2 = approximately 70 min), leveling off at 50% of the initial cell-associated HRP.
HRP appears to be transported relatively intact (approximately 50%) across rat alveolar epithelial barrier via nonspecific fluid-phase endocytosis. The transepithelial pinocytotic rate of alveolar epithelial cells is estimated to be about 25 nL/cm2/h.
评估辣根过氧化物酶（HRP，一种非特异性液相内吞标记物）通过在经组织培养处理的聚碳酸酯滤膜上生长的紧密（>2000欧姆·厘米²）大鼠肺泡上皮细胞单层体外模型的转运特性。
通过将HRP滴入供体液体后，根据受体液体中HRP的出现速率，作为供体[HRP]和温度的函数，估算单向HRP通量。在通量实验结束时，使用异硫氰酸荧光素（FITC）标记的HRP通过凝胶渗透色谱法测定两种浴液中存在的分子种类。测定转运实验结束时细胞相关的HRP活性，以及HRP的再循环和跨细胞移动速率。使用酶促测定法定量浴液和细胞中的HRP活性。
单向HRP通量是对称的，并且在供体[HRP]高达50微摩尔时呈线性增加。将温度从37℃降至4℃时，HRP的表观渗透系数降低了3.5倍。受体液体中存在的约50%的FITC标记物是完整的HRP。从顶端HRP孵育估计的细胞相关HRP比从基底外侧孵育的约大4倍。顶端孵育后细胞相关HRP再循环到顶端液体中迅速发生，半衰期（T1/2）约为5分钟，在初始细胞相关HRP的约67%处达到平台期，而HRP的跨细胞移动（进入基底外侧液体）的T1/2约为20分钟，在初始细胞相关HRP的约13%处达到稳态。HRP的基底外侧再循环也很快（T1/2约为5分钟），在初始基底外侧结合的HRP的约35%处达到稳态。基底外侧孵育后HRP的跨细胞移动较慢（T1/2约为70分钟），在初始细胞相关HRP的50%处趋于平稳。
HRP似乎通过非特异性液相内吞作用相对完整地（约50%）穿过大鼠肺泡上皮屏障。肺泡上皮细胞的跨上皮胞饮速率估计约为25纳升/平方厘米/小时。
