ABSTRACT
Physical-chemical and biological studies of hepatic bile suggest that biliary phospholipid molecules are secreted as unilamellar vesicles. Systematic ultrastructural studies of bile canaliculi were undertaken to visualize this event. Liver tissue was obtained from normal adult male rats (control), from bile salt-depleted rats (by overnight biliary diversion), and from depleted rats infused intravenously with a hydrophilic-hydrophobic congener series of common taurine-conjugated bile salts. Livers were fixed in situ either by modified chemical methods or by ultrarapid cryofixation. In control rats, chemical fixation revealed unilamellar vesicles 63 +/- 17 (+/- SD) nm in diameter, mostly free within canalicular lumena. Vesicles were infrequent in canaliculi of bile salt-depleted rats, but were present in canaliculi of rats infused with taurocholate. In cryofixed liver tissue, vesicles 67 +/- 13 nm in diameter were observed in canaliculi of control rats and bile-salt depleted rats infused with common bile salts. The majority of these vesicles were affixed to the luminal side of the canalicular membrane. The average number of vesicles per bile canaliculus was in agreement with that estimated on the basis of biliary phospholipid secretion rates, mean vesicle size, and area of close-packed phosphatidylcholine molecules. By immunoelectron microscopy, canalicular vesicles were free of actin and of a 100 kDa canalicular membrane protein. We conclude that biliary phospholipid molecules are secreted from hepatocytes into bile canalicular lumena as unilamellar vesicles approximately 63-67 nm in average diameter. We postulate that this secretion mechanism involves lumenal bile salt-induced vesiculation of lipid microdomains in the exoplasmic hemileaflet of the canalicular membrane.
Subject(s)
Bile Canaliculi/metabolism , Intracellular Membranes/ultrastructure , Lipid Metabolism , Liver/cytology , Animals , Bile Canaliculi/ultrastructure , Cryopreservation , Evaluation Studies as Topic , Fixatives , Liposomes , Liver/ultrastructure , Male , Microscopy, Electron , Microscopy, Immunoelectron , Rats , Rats, Sprague-DawleyABSTRACT
Employing microscope laser light-scattering spectroscopy, we investigated "primary" bile secretion into canalicular spaces of rat hepatocyte couplets in monolayer culture. Time-dependent scattered light intensities were fitted by bi-exponential decays. The "slow" decay was attributed to an undulating canalicular membrane motion, whereas the "fast" decay was consistent with rapidly diffusing intracanalicular vesicles with mean hydrodynamic radii (+/- SD) of 479 +/- 53 A. After addition of micromolar concentrations of common bile salts, increases in the amplitude of the fast component facilitated a quantitative estimate of vesicle secretion rates. A dose-response relationship with 0.1-200 microM sodium taurocholate was characterized by an initial concentration-dependent increase and then a decrease in the amplitude of the fast canalicular component. Since these taurocholate concentrations are nontoxic to cultured hepatocytes, the maximum in vesicle-sized particles at 10 microM taurocholate suggested that its critical micellar concentration of approximately 5 mM was attained within the canalicular spaces. Sodium taurolithocholate resulted in time- and dose-dependent diminution in vesicle secretion rates, which after 2 h was followed by spontaneous canalicular recovery. This suggested that acute bicellular "cholestasis" was followed by oxidative metabolism and detoxification of the monohydroxy bile salt. Microscope laser light-scattering spectroscopy should facilitate further physical-chemical and pathophysiological studies of bile secretion at the cellular level.
