Kinetics of uptake, transport, and accumulation of liposome-associated fluorescent dolichol esters in human fibroblasts

Abstract

The anthroyl and n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino (NBD) hexanoyl esters of dolichol were synthesized and incorporated into phospholipid liposomes. Fluorescence spectrometric methods were used to estimate the kinetics and dynamics of the incorporation and turnover of these esters in normal human fibroblasts. For anthroyl dolichol a saturable uptake of 2.5 × 103 pmol/106 fibroblasts was obtained. Half-maximum uptake was seen at a labeling concentration of 19 μM. The time required for half-maximum uptake of fluorescence (t1/2) was about 10 h. Over 50% of the anthroyl dolichol taken up remained in fibroblasts 24 h after the labeling medium was removed. Uptake was higher for esters of 9 isoprenes than for those with 16–21. Dolichol labeled with the NBD fluorophore appeared to enter fibroblasts in higher concentration than the same dolichol labeled with anthracene. Uptake was not influenced by the presence of agents that disrupt lysosome function (leupeptin and chloroquine) prior to or during fluorescence labeling. The amount of fluorescent dolichol in the (i) lysosomes and endosomes and (ii) nuclei of labeled fibroblasts was determined after Percoll density gradient centrifugation and cell lysis in culture, respectively. Most of the fluorescent dolichyl ester (and most of the free alcohol form) taken up by fibroblasts was recovered in lysosomes.Key words: anthroyl dolichol, n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino hexanoyl dolichol, lysosome, localization, laser scanning confocal microscopy, turnover.