Segregation of glucosylceramide and sphingomyelin occurs in the apical to basolateral transcytotic route in HepG2 cells

HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM), For this, the cells were labeled with fluorescent acyl chain-labeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]hexanoic acid (C-6-NBD) derivatives of either GlcCer (C-6-NBD-GlcCer) or SM (C-6-NBD-SM). The pool of the fluorescent lipid analogues present in the basolateral plasma membrane domain was subsequently depleted and the apically located C-6-NBD-lipid was chased at 37 degrees C. By using fluorescence microscopical analysis and a new assay that allows an accurate estimation of the fluorescent lipid pool in the apical membrane, qualitative and quantitative insight was obtained concerning kinetics, extent and (intra)cellular sites of the redistribution of apically located C-6-NBD-GlcCer and C6NBD-SM. It is demonstrated that both lipids display a preferential localization, C-6-NBD-GlcCer in the apical and C6NBD-SM in the basolateral area. Such a preference is expressed during transcytosis of both sphingolipids from the apical to the basolateral plasma membrane domain, a novel lipid trafficking route in HepG2 cells. Whereas the vast majority of the apically derived C-6-NBD-SM was rapidly transcytosed to the basolateral surface, most of the apically internalized C-6-NBD-GlcCer was efficiently redirected to the BC. The redirection of C-6-NBD-GlcCer did not involve trafficking via the Golgi apparatus. Evidence is provided which suggests the involvement of vesicular compartments, located subjacent to the apical plasma membrane. Interestingly, the observed difference in preferential localization of C-6-NBD-GlcCer and C-6-NBD-SM was perturbed by treatment of the cells with dibutyryl cAMP, a stable cAMP analogue. While the preferential apical localization of C-6-NBD-GlcCer was amplified, dibutyryl cAMP-treatment caused apically retrieved C-6-NBD-SM to be processed via a similar pathway as that of C-6-NBD-GlcCer. The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively, A role for non-Golgi-related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed