Ohtake Kazuo, Maeno Takaya, Ueda Hideo, Natsume Hideshi, Morimoto Yasunori
Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
Pharm Res. 2003 Feb;20(2):153-60. doi: 10.1023/a:1022485816755.
The purpose of this work was to characterize the main transport pathway of hydrophilic macromolecules induced by poly-L-arginine (poly-L-Arg; molecular weight 42.4 kDa) across the excised rabbit nasal epithelium.
Excised rabbit nasal epithelium was mounted in an Ussing-type chamber for measurement of fluorescein isothiocyanate-labeled dextran (FD-4; molecular weight 4.4 kDa) transport and transepithelial electrical resistance (TEER). The main transport pathway of FD-4 enhanced by poly-L-Arg was evaluated using confocal laser scanning microscopy. Immunolocalization of junction proteins (ZO-1, occludin, and E-cadherin) after treatment with poly-L-Arg was also observed.
After apical application of a poly-L-Arg (0.05, 0.5, and 5 mg/mL), the permeability coefficient of FD-4 increased by 1.6-, 2.9-, and 5.2-fold, respectively, compared with the control of 5.2 +/- 1.3 x 10(-7) cm/s. Consistent with the increase in transport, there was a concurrent reduction in TEER. At a concentration of 0.05 mg/mL poly-L-Arg. both FD-4 transport and TEER returned to the control level. A good correlation was obtained between the FD-4 permeability coefficient and 1/TEER. Basolateral application of poly-L-Arg at 5 mg/mL, however, did not increase FD-4 transport. Marked FD-4 fluorescence was located in the paracellular spaces after treatment with apical poly-L-Arg compared with that in the absence of poly-L-Arg. Immunofluorescence of ZO-1, occludin, and E-cadherin in cell-to-cell junctions was reduced and distributed into the cytoplasm by apical application of poly-L-Arg, suggesting that poly-L-Arg regulates the junction proteins to enhance paracellular permeability across the nasal epithelium. After pretreatment with either 2,4-dinitrophenol or ouabain, the enhancing effect of apical poly-L-Arg was abolished, indicating the contribution of metabolic energy (cell viability) to the poly-L-Arg-mediated enhancing effect.
In the nasal epithelium, apical poly-L-Arg appears to increase predominantly the paracellular transport of hydrophilic macromolecules via disorganization of tight- and adherens-junction proteins. The regulatory mechanism of the poly-L-Arg effect is likely to be dependent on energy-requiring cellular processes.
本研究旨在表征聚-L-精氨酸（聚-L-Arg；分子量42.4 kDa）诱导亲水性大分子跨切除的兔鼻上皮的主要转运途径。
将切除的兔鼻上皮安装在Ussing型小室中，用于测量异硫氰酸荧光素标记的葡聚糖（FD-4；分子量4.4 kDa）的转运和跨上皮电阻（TEER）。使用共聚焦激光扫描显微镜评估聚-L-Arg增强的FD-4的主要转运途径。还观察了聚-L-Arg处理后连接蛋白（ZO-1、闭合蛋白和E-钙黏蛋白）的免疫定位。
在顶端应用聚-L-Arg（0.05、0.5和5 mg/mL）后，与对照组5.2±1.3×10⁻⁷ cm/s相比，FD-4的渗透系数分别增加了1.6倍、2.9倍和5.2倍。与转运增加一致，TEER同时降低。在聚-L-Arg浓度为0.05 mg/mL时，FD-4转运和TEER均恢复到对照水平。FD-4渗透系数与1/TEER之间具有良好的相关性。然而，在基底外侧应用5 mg/mL的聚-L-Arg并没有增加FD-4的转运。与未使用聚-L-Arg相比，顶端应用聚-L-Arg处理后，明显的FD-4荧光位于细胞旁间隙。顶端应用聚-L-Arg后，细胞间连接处的ZO-1、闭合蛋白和E-钙黏蛋白的免疫荧光减少并分布到细胞质中，表明聚-L-Arg调节连接蛋白以增强鼻上皮的细胞旁通透性。用2,4-二硝基苯酚或哇巴因预处理后，顶端聚-L-Arg的增强作用被消除，表明代谢能量（细胞活力）对聚-L-Arg介导的增强作用有贡献。
在鼻上皮中，顶端聚-L-Arg似乎主要通过紧密连接和黏附连接蛋白的紊乱增加亲水性大分子的细胞旁转运。聚-L-Arg作用的调节机制可能依赖于需要能量的细胞过程。
