INTRACELLULAR SITES INVOLVED IN THE BIOGENESIS OF BILE CANALICULI IN HEPATIC CELLS

Studies in hepatoma cells and hepatocytes have revealed that the bioge nesis of bile canalicular membrane involves microvilli-lined vesicles (MLV), which are formed in well diferentiated cells. The vesicles grow as a function of time and are presumably vectorially transported to c ell surface contact sites of attached cells. We demonstrate that a flu orescent head group-labeled lipid analog, N-(lissamine rhodamine B sul fonyl)phosphatidylethanolamine (N-Rh-PE), after its exogenous insertio n into the plasma membrane of HepG2 cells at 40 degrees C, accumulates in these microvilli-lined vesicles at 37 degrees C. This shows that t he MLV are a target for plasma membrane-derived lipids. Furthermore, a lso the Gels apparatus is involved in the formation of the vesicles. A fter initial accumulation of the fluorescent sphingolipid precursor, 6 -[N-(7-nitrobenz-2 oxa-1,3 diazol 4-yl)amino]hexanoic acid (C-6-NBD)-c eramide in the Golgi apparatus at 37 degrees C, prolonged incubation a t 37 degrees C results in the appearance of NBD fluorescence in the mi crovilli-lined vesicles. The transport route for the Golgi-derived mat erial to the developing bile canalicular vesicle is not an indirect pa thway, i.e. involving transcytosis via the basolateral plasma membrane . This could be demonstrated by including bovine serum albumin (BSA) i n the incubation media, a lipid scavenger that will remove any C-6-NBD -lipids exposed at the basolateral membrane. At these conditions, lipi d trafficking between the Golgi complex and MLV still occurred. We fur ther demonstrate that the targeting from the Golgi apparatus to the bi le canaliculus is also operational in isolated human hepatocytes. Tire latter result suggests that the Golgi complex is involved in both the formation of bile canaliculi and in bile secretion in fully different iated cells.