Abstract
During bile formation by the liver, large volumes of water are transported across two epithelial barriers consisting of hepatocytes and cholangiocytes (i.e. intrahepatic bile duct epithelial cells). We recently reported that a water channel, aquaporin-channel-forming integral protein of 28 kDa, is present in cholangiocytes and suggested that it plays a major role in water transport by these cells. Since the mechanisms of water transport across hepatocytes remain obscure, we performed physiological, molecular, and biochemical studies on hepatocytes to determine if they also contain water channels. Water permeability was studied by exposing isolated rat hepatocytes to buffers of different osmolarity and measuring cell volume by quantitative phase contrast, fluorescence and laser scanning confocal microscopy. Using this method, hepatocytes exposed to hypotonic buffers at 23 degrees C increased their cell volume in a time and osmolarity-dependent manner with an osmotic water permeability coefficient of 66.4 x 10(-4) cm/s. In studies done at 10 degrees C, the osmotic water permeability coefficient decreased by 55% (p < 0.001, at 23 degrees C; t test). The derived activation energy from these studies was 12.8 kcal/mol. After incubation of hepatocytes with amphotericin B at 10 degrees C, the osmotic water permeability coefficient increased by 198% (p < 0.001) and the activation energy value decreased to 3.6 kcal/mol, consistent with the insertion of artificial water channels into the hepatocyte plasma membrane. Reverse transcriptase polymerase chain reaction with hepatocyte RNA as template did not produce cDNAs for three of the known water channels. Both the cholesterol content and the cholesterol/phospholipid ratio of hepatocyte plasma membranes were significantly (p < 0.005) less than those of cholangiocytes; membrane fluidity of hepatocytes estimated by measuring steady-state anisotropy was higher than that of cholangiocytes. Our data suggests that the osmotic flow of water across hepatocyte membranes occurs mainly by diffusion via the lipid bilayer (not by permeation through water channels as in cholangiocytes).
Highlights
Bile formation by the liver involves two phases: secretion of primary bile by hepatocytes at the canalicular domain and delivery to a network of interconnecting ducts where bile is modified by cholangiocytes via the secretion of ions and water
By quantitative phase contrast microscopy, individual hepatocytes exposed to hypotonic (30 mosM) buffers at 23 °C increased their cell diameter by 16% (i.e. a 55% increase in cell volume) 10 s after exposure (Fig. 1A)
Our data suggest that the osmotic flow of water across the hepatocyte membranes occurs mainly by diffusion across the lipid bilayer rather than by permeation through water channels, a process we have previously described in cholangiocytes (13)
Summary
Bile formation by the liver involves two phases: secretion of primary bile by hepatocytes at the canalicular domain and delivery to a network of interconnecting ducts where bile is modified by cholangiocytes via the secretion of ions and water. We reported that cholangiocytes express the message and protein for aquaporin-CHIP and proposed that cholangiocytes transport water in a bidirectional fashion via a channel-mediated pathway (13) which likely accounts for the absorptive and secretory modification of ductal bile by cholangiocytes. To extend these studies to the level of primary bile secretion and broaden our understanding of water movement in hepatic epithelia, we performed both direct and comparative functional, molecular, and biochemical studies to determine the molecular mechanisms by which hepatocytes transport water
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