Isolated rat hepatocyte couplets in short-term culture: Structural characteristics and plasma membrane reorganization

Abstract

Studies of canalicular bile secretion have been limited due to lack of direct access to the canalicular lumen. Isolated rat hepatocyte couplets, consisting of two hepatocytes enclosing a canalicular space, have been proposed as a primary secretory unit that may be useful for direct studies of unmodified canalicular bile secretion. The present study examines their structural characteristics and plasma membrane reorganization. The canalicular space of freshly isolated hepatocyte couplets is freely permeable to ruthenium red, but within 4 hr the junctional borders reseal in most couplets which then exclude ruthenium red from the luminal area. These resealed spaces expand in 61.8 ± 10% of couplets as secretion is elaborated and after 4 hr in monolayer culture, 12.7 ± 4.7% of the canalicular spaces are dilated to diameters greater than 3 μ. Normal-appearing microvilli line these canalicular membranes in the majority of dilated spaces as assessed by electron microscopy. Immediately after isolation, Mg⁺⁺-ATPase, a histochemical marker for canalicular membranes, is located as a discrete band corresponding to the normal in vivo circumferential distribution of the canalicular membrane domain. However, this pattern of Mg⁺⁺-ATP-ase staining rapidly diminishes and reorganizes at the remaining canalicular pole within several hours. This membrane reorganization is a microfilament-dependent process, since it is inhibited by cytochalasin D but not by colchicine, an inhibitor of microtubular function or cycloheximide, an inhibitor of new protein synthesis. Thus, 3 to 4 hr after isolation, the isolated hepatocyte couplet has reorganized its canalicular membrane, retaining polarity of its excretory domain only at the remaining junctional contacts between adjacent hepatocytes. Secretion is elaborated into the ruthenium red impermeant canalicular lumen permitting direct studies of canalicular bile secretion by micropuncture, electrophysiologic and quantitative microscopic techniques.