Permeabilized hepatocyte couplets. Adenosine triphosphate-dependent bile canalicular contractions and a circumferential pericanalicular microfilament belt demonstrated

Abstract

The motility of bile canaliculi was examined in hepatocyte couplets permeabilized with palmitoyl lysophosphatidyl choline in a dosage regimen that drastically affected secretory function, yet maintained relative integrity of the cellular cytoskeleton. The permeabilized cells showed no exclusion of trypan blue, notable cytoplasmic organelle and membrane damage, and no uptake or secretion of either fluorescein diacetate or sodium fluorescein. However, bile canalicular structure remained relatively intact and actin and myosin were localized immunocytochemically in the pericanalicular region. Coincident with the administration of 1 mM ATP, 2 mM Mg2+, and 1 microM Ca2+, the canaliculi contracted with partial or complete luminal closure. ADP, AMP, or AMP-PCP could not be substituted for ATP. A dose-dependent relationship was shown between ATP concentration and canalicular contraction rate. The permeabilization procedure also provided enhanced visualization of pericanalicular microfilaments, believed to be actin filaments, and their organization into two layers: an inner membrane-associated network, and an outer filament bundle that inserted into belt junctions (zonulae adherentes). The organization of the microfilament belt of contiguous hepatocytes was such that it formed a circumferential band of microfilaments around the canaliculus. It is analogous to contractile filament belts found in the apical terminal web region of other epithelia. It was also observed that with canalicular luminal closure, there was a change in the organization of the pericanalicular microfilaments. It is concluded that in hepatocyte couplets, differential sensitivity of cell components to permeabilization can be achieved with palmitoyl lysophosphatidyl choline. In addition, the results provide evidence that the bile canaliculus has the capacity to be a contractile structure even in the absence of secretion, that canalicular contraction is ATP-dependent, and hence is a dynamic process.