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Vol. 9, Issue 7, 1939-1949, July 1998
Department of Physiological Chemistry, Faculty of Medical Sciences,
University of Groningen, 9713 AV Groningen, The Netherlands
In polarized HepG2 hepatoma cells, sphingolipids are transported to
the apical, bile canalicular membrane by two different transport
routes, as revealed with fluorescently tagged sphingolipid analogs. One
route involves direct, transcytosis-independent transport of
Golgi-derived glucosylceramide and sphingomyelin, whereas the other
involves basolateral to apical transcytosis of both sphingolipids. We
show that these distinct routes display a different sensitivity toward
nocodazole and cytochalasin D, implying a specific transport dependence
on either microtubules or actin filaments, respectively. Thus,
nocodazole strongly inhibited the direct route, whereas sphingolipid
transport by transcytosis was hardly affected. Moreover, nocodazole
blocked "hyperpolarization," i.e., the enlargement of the apical
membrane surface, which is induced by treating cells with
dibutyryl-cAMP. By contrast, the transcytotic route but not the direct
route was inhibited by cytochalasin D. The actin-dependent step during
transcytotic lipid transport probably occurs at an early endocytic
event at the basolateral plasma membrane, because total lipid uptake
and fluid phase endocytosis of horseradish peroxidase from this
membrane were inhibited by cytochalasin D as well. In summary, the
results show that the two sphingolipid transport pathways to the apical
membrane must have a different requirement for cytoskeletal elements.
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