| 9512098 |
Takakura Y, Oka Y, Hashida M: Cellular uptake properties of oligonucleotides in LLC-PK1 renal epithelial cells. Antisense Nucleic Acid Drug Dev. 1998 Feb;8(1):67-73.
The objective of this study was to clarify the renal uptake characteristics of oligonucleotides at a cellular level using LLC-PK1 renal epithelial cells derived from the proximal tubule. The association of [35S]-labeled 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides with the monolayers of polarized LLC-PK1 cells cultured on polycarbonate filter was characterized after apical or basolateral application. The cellular association of PO and PS at both apical and basolateral membranes was time dependent and temperature dependent, and the apparent association amount of PS was larger than that of PO. The PO and PS association after apical application was saturable, with the apparent Km and Vmax values determined to be 5.4 microM and 0.14 nmol/mg protein for PO and 0.22 microM and 0.11 nmol/mg protein for PS, respectively. In contrast, almost linear kinetics were observed after basolateral application within a tested concentration range. The association was inhibited significantly by sodium azide and chloroquine, suggesting that an energy-dependent endocytotic process was involved. Internalization and subsequent transport to endosome and lysosome compartments of FITC-labeled oligonucleotides were shown by confocal laser scanning microscopy. The present study has demonstrated that both types of oligonucleotides are taken up by LLC-PK1 cells from both apical and basolateral surfaces probably via an endocytosis mechanism. |
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