Bile canalicular contraction is coincident with reorganization of pericanalicular filaments and co-localization of actin and myosin-II.

We examined the relationships between actin-myosin interaction and bile canalicular contraction using a new experimental model: cytoskeleton-enriched canalicular membranes (CCM). In CCM, the bile canaliculus compartment is isolated complete with membrane-attached pericanalicular actin filaments and the surrounding intermediate filament sheath. Immunofluorescence and immunoelectron microscopy showed that actin and myosin-II were distributed over pericanalicular microfilaments that insert into adherens (belt) junctions; intermediate filaments predominantly inserted into desmosomes. The addition of "contraction solution" (1 microM Ca2+, 1 mM ATP) resulted in closure of CCM lumens, which was interpreted as canalicular contraction. Contraction was also associated with shortening and/or twisting of canaliculi. Rearrangement of actin filaments and myosin-II with co-localization of actin and myosin was observed. Evidence is also provided for attachment of actin-myosin-II aggregates to intermediate filaments coincident with contraction, suggesting a key scaffold function for intermediate filaments of the canaliculus. Attention is drawn to the overall similarity of structure-function dynamics in hepatic apical membranes to those described in intestinal brush border membrane preparations. The results are consistent with dynamic actin-myosin interaction with co-localization of actin and myosin-II in filament clumps coincident with canalicular contraction.

Elemental changes in hepatocyte couplet cell cultures following treatment with vasopressin

Hepatocyte couplet cell cultures have been used as a cell model to study the contractile nature of the bile canaliculus. It is generally accepted that a rise in cytoplasmic Ca activates the actomyosin ATPase resulting in the contraction of the pericanalicular actomyosin complex and the closure of the bile canaliculus. Vasopressin, a Ca mobilizing hormone, has been shown to increase the cytoplasmic Ca and induce bile canalicular contraction.In the present study rat hepatocyte couplet cultures were prepared as described by Oshio and Phillips. After 4 hours the volume of the culture media was decreased and the cells were liberated from the culture surface with a rubber policeman. Cells were then treated with vasopressin at a concentration of 10-9 M for time periods of 0, 30, 60,90, 120, 180, 240 and 300 seconds and drops of cell suspension were frozen by either slam freezing them on a liquid nitrogen cooled, highly polished copper anvil or plunge freezing them into liquid nitrogen cooled ethane.