Role of cytoskeleton in canalicular contraction in cultured differentiated hepatocytes.

Abstract

We and others have published data that indicate that the role played by microtubules and microfilaments in biliary secretion is as follows: microtubules play a part in secretion and microfilaments play a part in the canalicular contraction. To further study the role of the cytoskeleton in canalicular contraction, we observed the contraction of bile canaliculi (BC) induced by vasopressin (VP) in cultured differentiated hepatocytes treated with several agents that selectively rearrange the cytoskeleton. The hepatocytes obtained from 14-day-old rats were cultured for 48 hours. The BC formed between the cells were dilated and closely sealed by junctional complexes. Ruthenium red stain showed that the junctional complexes of the BC were tightly sealed. Cytoskeletal changes were observed by double-labeled fluorescence microscopy. A spontaneous contraction of the BC was rarely seen during a 60-minute observation period in controls. When the hepatocytes were incubated with VP (10(-8) M), the canalicular contraction began at 30 minutes, gradually progressed, and was complete by 60 minutes. The contraction was reversed after 60 minutes of incubation in VP-free medium. In cytochalasin B-treated hepatocytes, actin appeared to form pools around the dilated BC, and the canalicular contraction after VP was rarely observed. In colchicine-treated hepatocytes, the microtubules were depolymerized. Although the BC appeared unaffected by colchicine alone, the canalicular contraction induced by VP was markedly decreased. beta-lumicolchicine had no effect on the cytoskeleton or on the canalicular contraction. Mg2(+)-ATPase histochemistry revealed that the BC that did not contract after VP contained little Mg2(+)-ATPase reaction product. When the BC contracted, diverticula came off to form diacytotic vesicles, as indicated by the presence of the BC marker enzyme reaction product within the vesicles. Colchicine treatment blocked the diacytotic process. This prevented the contraction stimulated by VP, because all of the routes of escape of the canalicular contents were blocked off, including diacytosis. In conclusion, the integrity of actin filaments and Mg2(+)-ATPase is necessary for VP-induced contraction, and the integrity of microtubules is essential for regurgitation of BC content (diacytosis).