Active microtubule-dependent secretion of a fluorescent bile salt derivative in skate hepatocyte clusters

Fluorescence microscopy and video image analysis were used to study the transport of a fluorescent bile acid derivative [N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oyl-2-aminoethanesulfonate (NBD-TC)] in isolated clusters of hepatocytes from the little skate Raja erinacea. Analysis of images of hepatocyte clusters that were incubated in medium with 0.5-1 microM NBD-TC showed that the fluorescent derivative accumulated in the cells and that the clusters retained a patent canalicular lumen as well as the ability to actively transport the bile acid derivative from the cells into the lumen; i.e., the lumen-to-cell fluorescence ratio greatly exceeded unity. NBD-TC uptake by hepatocytes was inhibited by several organic anions, of which taurocholate was the most effective. Uptake was also blocked by metabolic inhibitors and by incubation in the cold. Neither Na replacement nor increased medium K, which depolarizes the membrane electrical potential [potential d...