Rat Hepatocytes Transport Water Mainly via a Non-channel-mediated Pathway

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°C increased their cell volume in a time and osmolarity-dependent manner with an osmotic water permeability coefficient of 66.4 ؋ 10 ؊4 cm/s. In studies done at 10°C, the osmotic water permeability coefficient decreased by 55% (p < 0.001, at 23°C; t test). The derived activation energy from these studies was 12.8 kcal/mol. After incubation of hepatocytes with amphotericin B at 10°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).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.Bile secretion by hepatocytes involves the active transport of both organic and inorganic solutes, followed by the passive movement of water into bile canaliculi in response to osmotic gradients established by these solutes (1). While a substantial amount of recent data have permitted a clearer understanding of the cellular mechanisms regulating solute transport by hepatocytes (2, 3), the mechanisms regulating water movement across hepatocytes remain obscure (2, 3).Theoretically, water may move across the epithelial barrier of hepatocytes by two pathways: a paracellular pathway between adjacent cells, or a transcellular pathway across the cell (4). Further, transcellular water movement may occur throu...

Expression of aquaporin-4 water channels in rat cholangiocytes

“…Thus, although RT-PCR identified a full length and an alternatively spliced form of AQP4 using RNA extracted from total rat liver, 11 other studies could not detect AQP4 using the RNase protection assay or immunoblotting analysis. 17 Those studies, together with our previous data showing no expression of AQP4 in purified hepatocytes, 10 suggested to us that, if AQP4 was present in liver, it was likely localized to a minority cell population such as cholangiocytes (i.e., 3%-5% of the total liver cell population). Therefore, in the present study, we used material derived directly from highly purified cholangiocytes, an approach that consistently showed AQP4 expression at both mRNA and protein levels.…”

“…Hepatocytes (Ͼ98% pure) were isolated from livers of male Fischer rats by collagenase perfusion and mechanical disruption as described previously. 10 These cells were snap-frozen and stored at Ϫ70°C until used for RNA isolation or for the preparation of plasma membranes. Hepatocytes do not express AQP4, 10 and for this reason they were used as a negative control in our studies.…”

“…The size of cholangiocytes was determined by quantitative phase contrast microscopy, a methodology previously validated by us. 10 Briefly, serial photographs of the same group of cells placed on coverslips coated with polylysine, in isotonic (300 mOsm) and hypotonic media (30 mOsm) were digitized and cell diameters measured with an image analysis software program. Cell volumes were estimated based on the spherical shape of cholangiocytes using 4.5 µm immunomagnetic beads as internal standards.…”

The Water Channel Aquaporin-8 Is Mainly Intracellular in Rat Hepatocytes, and Its Plasma Membrane Insertion Is Stimulated by Cyclic AMP

“…We previously found based on biophysical and molecular biology studies that water transport across hepatocyte plasma membranes occurs mainly via a non-channel mediated pathway (6). As this observation seems to be in contradiction with the recent identification of transcript for the water channel aquaporin-8 (AQP8) 1 in hepatocytes (7-9), we further explored this issue by studying the protein expression, subcellular localization, and possible regulation of AQP8 water channels in isolated rat hepatocytes.…”

Cryopreservation of Hepatocytes for Bioartificial Liver Devices

“…Furthermore, the cells are biochemically very complex, and retention of their liver-specific functions in culture post-thaw is a difficult task. Finally, the large size and slow water transport characteristics (Yano et al, 1996) of the hepatocyte may be another reason why they are difficult to cryopreserve compared with other cell types.…”