Wielinga P R, de Waal E, Westerhoff H V, Lankelma J
Academisch Ziekenhuis Vrije Universiteit, Department of Medical Oncology, P.O. Box 7057, Room BR232, 1007 MB Amsterdam, The Netherlands.
J Pharm Sci. 1999 Dec;88(12):1340-7. doi: 10.1021/js980497z.
Studies on transcellular transport across epithelial cell layers are performed mostly by discontinuous sampling of the transported compound. This has several drawbacks, e.g., it gives disturbances in volume, it limits the time-resolution, and is often laborious. In this report we introduce a method to measure transepithelial transport of fluorescent compounds continuously. The time-resolution is at the (sub)minute scale, allowing the measurement of the change in transport rate before and after transport modulation. We will describe how we used the method to measure transcellular and paracellular transport. For highly membrane-impermeable compounds, the paracellular transport and the regulation of the tight junctions was studied in wild-type and MDR1 cDNA transfected epithelial canine kidney cells (MDCKII). The effect of the multidrug transporter P-glycoprotein (Pgp) on the transepithelial transport was studied. Addition of the Pgp inhibitor SDZ PSC 833 showed a modulation of the idarubicin (IDA) and daunorubicin (DNR) transport, which was larger during transport from the basolateral to the apical side than in the reverse direction. By modeling the transepithelial transport, we found that in these cells Pgp had more effect on the basolateral to apical transport than vice versa, which can be attributed to a relatively large passive permeation coefficient for the cellular basolateral plasma membrane.
关于跨上皮细胞层的跨细胞转运研究大多是通过对转运化合物进行间断采样来进行的。这有几个缺点，例如，它会导致体积干扰，限制时间分辨率，并且通常很费力。在本报告中，我们介绍一种连续测量荧光化合物跨上皮转运的方法。时间分辨率处于（亚）分钟尺度，能够测量转运调节前后转运速率的变化。我们将描述如何使用该方法来测量跨细胞转运和细胞旁转运。对于高度膜不透性的化合物，在野生型和转染了MDR1 cDNA的上皮犬肾细胞（MDCKII）中研究了细胞旁转运和紧密连接的调节。研究了多药转运蛋白P-糖蛋白（Pgp）对跨上皮转运的影响。添加Pgp抑制剂SDZ PSC 833显示对伊达比星（IDA）和柔红霉素（DNR）的转运有调节作用，从基底外侧到顶端侧的转运过程中的调节作用比反向转运时更大。通过对跨上皮转运进行建模，我们发现在这些细胞中，Pgp对从基底外侧到顶端的转运的影响比对反向转运的影响更大，这可归因于细胞基底外侧质膜相对较大的被动渗透系数。
