RAT HEPATOCYTES TRANSPORT WATER MAINLY VIA A NON-CHANNEL-MEDIATED PATHWAY

During bile formation by the liver, large volumes of water are transpo rted across two epithelial barriers consisting of hepatocytes and chol angiocytes (i.e. intrahepatic bile duct epithelial cells), Tire recent ly reported that a water channel, aquaporin-channel-forming integral p rotein of 28 kDa, is present in cholangiocytes and suggested that it p lays a major role in water transport by these cells, Since the mechani sms of water transport across hepatocytes remain obscure, we performed physiological, molecular, and biochemical studies on hepatocytes to d etermine if they also contain water channels. Water permeability was s tudied by exposing isolated rat hepatocytes to buffers of different os molarity and measuring cell volume by quantitative phase contrast, flu orescence and laser scanning confocal microscopy, Using this method, h epatocytes exposed to hypotonic buffers at 23 degrees C increased thei r cell volume in a time and osmolarity-dependent manner with an osmoti c water permeability coefficient of 66.4 x 10(-4) cm/s, In studies don e at 10 degrees C, the osmotic water permeability coefficient decrease d 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 hepat ocytes with amphotericin B at 10 degrees C, the osmotic water permeabi lity coefficient increased by 198% (p < 0.001) and the activation ener gy value decreased to 3.6 kcal/mol, consistent with the insertion of a rtificial water channels into the hepatocyte plasma membrane, Reverse transcriptase polymerase chain reaction with hepatocyte RNA as templat e did not produce cDNAs for three of the known mater channels. Both th e cholesterol content and the cholesterol/phospholipid ratio of hepato cyte plasma membranes were significantly (p < 0.005) less than those o f cholangiocytes; membrane fluidity of hepatocytes estimated by measur ing steady-state anisotropy was higher than that of cholangiocytes. Ou r data suggests that the osmotic flow of water across hepatocyte membr anes occurs mainly by diffusion via the lipid bilayer (not by permeati on through water channels as in cholangiocytes).