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Cholangiocytes Express the Aquaporin CHIP and Transport Water Via a Channel-Mediated Mechanism STUART K

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Cholangiocytes Express the Aquaporin CHIP and Transport Water Via a Channel-Mediated Mechanism STUART K Proc. Natl. Acad. Sci. USA Vol. 91, pp. 13009-13013, December 1994 Physiology Cholangiocytes express the aquaporin CHIP and transport water via a channel-mediated mechanism STUART K. ROBERTS*, MOTOYOSHI YANO*, YOSHIYUKI UENO*, LINH PHAM*, GIANFRANCO ALPINI*, PETER AGREt, AND NICHOLAS F. LARUSSO*t Departments of *Internal Medicine and tBiochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905; and tDepartments of Biological Chemistry and Medicine, Johns Hopkins University, Baltimore, MD 21205 Communicated by Ralph T. Holman, August 15, 1994 (receivedfor review April 6, 1994) ABSTRACT Cholangiocytes line the intrahepatic bile pore capable of transporting water across the plasma mem- ducts and regulate salt and water secretion during bile forma- brane in a rapid, relatively temperature-independent and tion, but the mechanism(s) regulating ductal water movement mercury-sensitive manner. Moreover, immunohistochemical remains obscure. A water-selective channel, the aquaporin (13-16) and Northern blot (10, 17, 18) analyses and in situ CHIP, was recently described in several epithelia, so we tested hybridization (19) have demonstrated that CHIP has a wide the hypothesis that osmotic water movement by cholangiocytes tissue distribution, suggesting that it might be a general water is mediated by CHIP. Isolated rodent cholangiocytes showed a channel (9). Thus, we began to examine the mechanism(s) by rapid increase in volume in the presence of hypotonic extra- which water traverses biliary epithelia. cellular buffers; the ratio ofosmotic to diffusional permeability coefficients was >10. The osmotically induced increase in MATERIALS AND METHODS cholangiocyte volume was inversely proportional to buffer Cholangiocytes. Cholangiocytes [>95% pure by specific osmolality, independent of temperature, and reversibly markers (20)] were isolated from livers of male Fiseher rats blocked by HgCl2. Also, the luminal area of isolated, enclosed (21). For flow cytometry, cholangiocytes were serially incu- bile duct units increased after exposure to hypotonic buffer and bated with (i) 20% normal goat serum, (ii) a 1:3 dilution of a was reversibly inhibited by HgCl2. RNase protection assays, mouse monoclonal antibody specific for cholangiocytes (21), anti-CHIP immunoblots, and immunocytochemistry con- and (iii) a 1:100 dilution of polyclonal goat anti-mouse IgM firmed that CHIP transcript and protein were present in conjugated to fluorescein isothiocyanate (FITC; Southern isolated cholangiocytes but not in hepatocytes. These results Biotechnology Associates) and identified by their increased demonstrate that (0) isolated cholangiocytes and intact, polar- FITC fluorescence compared with negative control cells, ized bile duct units manifest rapid, mercury-sensitive increases prepared without incubation with the monoclonal antibody. in cell size and luminal area, respectively, in response to Over 91% of cells with increased FITC fluorescence were osmotic gradients and (it) isolated cholangiocytes express aqua- positive for cholangiocyte-specific markers (20). Cell viabil- porin CHIP at both the mRNA and the protein level. The data ity was determined by trypan blue exclusion. implicate aquaporin water channels in the transcellular move- Hepatocytes. Hepatocytes (>97% pure by morphological ment of water across cholangiocytes lining intrahepatic bile appearance) were isolated as described (22). ducts and provide a plausible molecular explanation for ductal Bile Duct Units. Enclosed polarized bile duct units (BDUs) water secretion. were prepared from livers of male rats (4). Quantitative Phase-Contrast Microscopy. The size of cho- Bile formation by the liver involves secretion of bile by langiocytes in extracellular buffers was measured with an hepatocytes and delivery to a network of interconnecting inverted phase-contrast microscope. Freshly isolated cho- ducts where bile is modified by cholangiocytes, the epithelial langiocytes were mounted on a microscope stage in isotonic cells that line these conduits inside the liver. Bile secretion by (300 mOsm) Hepes-buffered saline (HBS: 140 mM NaCl/5.4 cholangiocytes contributes to total bile flow through the mM KCl/0.8 mM Na2HPO4/25 mM Na Hepes/0.8 mM spontaneous and agonist-induced secretion of both ions and MgSO4, pH 7.4, 22°C). Cells were exposed to extracellular water (1). While data have been accumulating on the cellular buffers ofdiffering osmolality (range, 30-300 mOsm); buffers mechanisms regulating ion transport by cholangiocytes (2-4), were prepared by diluting HBS with the appropriate volume the mechanisms regulating water movement across biliary of distilled water. Serial photographs were taken and cell epithelia remain undefined (5, 6). diameters were measured in a randomized, blinded manner Conceptually, water may move across biliary epithelia by from projected images by using the 4.5-,m immunomagnetic two pathways: a paracellular pathway between cholangio- beads as internal standards. Cell volumes were then derived cytes or a transcellular pathway across both the apical and based on the spherical shape of freshly isolated cholangio- basolateral cholangiocyte plasma membranes (5, 7). Further, cytes (21); results are expressed as percent change in cell transcellular water movement may occur by simple diffusion volume over time. The osmotic water permeability coeffi- across the lipid bilayer or through discrete membrane pro- cient (Pf, cm/sec) was calculated from osmotic swelling data, teins that form water channels (8). A family of membrane initial cholangiocyte volume (V0 = 1.56 x 10-9 cm3), and water channels, referred to as aquaporins, was recently surface area (S = 6.52 x 10-6 cm2) (23). identified (9). The aquaporin CHIP [ghannel-forming integral Similarly, enclosed polarized BDUs were exposed to bath- membrane protein of 28 kDa] is the first characterized ing buffers of differing osmolality and the time-dependent molecular water channel (10). When expressed in Xenopus change in the luminal area was measured from serial photo- laevis oocytes (11) or reconstituted into proteoliposomes graphs by the point-counting method (24). (12), CHIP behaves as an osmotically driven, water-selective Flow Cytometry. The size of cholangiocytes was also assessed by flow cytometry on a dual-laser flow cytometer The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: FITC, fluorescein isothiocyanate; BDU, intrahepatic in accordance with 18 U.S.C. §1734 solely to indicate this fact. bile duct unit; RT, reverse transcription. 13009 Downloaded by guest on October 4, 2021 13010 Physiology: Roberts et al. Proc. Natl. Acad. Sci. USA 91 (1994) (25). FITC fluorescence and 100 incident light scatter of cells A were measured (26) and data were LYSYS II analyzed by Isotonic software (Becton Dickinson). (300 mOsm) FITC-labeled cholangiocytes were suspended in isotonic HBS and mounted on the flow cytometer. After baseline values were obtained, cells were exposed to extracellular Hypotonic buffers of 30-300 mOsm and light scatter was measured over (30 mOsm) the next 30 sec; results are expressed as percent change in light scatter over time. Osec 30sec Diffusional Water Permeability Studies. The diffusion per- meability coefficient of rodent cholangiocytes was deter- B 180 mined experimentally by the linear diffusion technique (27). Aquaporin CHIP Gene Expression. Total was 160- 30 mOsm cellular RNA 100 mOsm isolated from whole organs and pure preparations of cholan- giocytes and hepatocytes (28). ' 1401 Reverse Transcription-Polymerase Chain Reaction (RT- c,O)- 120 l OmOsm PCR). Specific oligonucleotide DNA primers were based on the rat CHIP DNA sequence (17). With these primers and 100- m n total cellular RNA as template, cDNA was generated by 0 RT-PCR and sequenced (29). 5 10 15 20 25 30 RNase Protection Assay. A 279-bp cDNA corresponding to C 110 Time (sec) nt 215-494 of the cDNA encoding rat CHIP was prepared by 100 _00mT m RT-PCR using total RNA from rat cholangiocytes as tem- 909 o D0- 200 mOsm plate. This cDNA was cloned into the pCR II vector (Invit- 80 1 rogen), and an antisense RNA was transcribed from pCR II 70. as described (30). RNase protection assays were performed )m60100mOsmm (30) with the CHIP antisense RNA probe and total RNA from pure preparations of cholangiocytes and hepatocytes. 30 mOsm 40 Aquaporin CHIP Protein Expression. Immunoblotting (13) 0 0 5 10 5 20 25 used a polyclonal anti-CHIP antibody (13) and protein ex- 30 tracted from membranes prepared from human erythrocytes Time (sec) and rat liver cells (31, 32). Immunocytochemistry with prep- FIG. 1. Osmotic water transport in rodent cholangiocytes. (A) arations of isolated liver cells (21) used an affinity-purified Phase-contrast micrographs of purified cholangiocytes in isotonic anti-CHIP antibody (0.3 ,g/ml) (13) and a Vectastain ABC (Upper) and hypotonic (30 mOsm) (Lower) buffers. Note the immu- kit (Vector Laboratories). Staining specificity was confirmed nomagnetic beads attached to cholangiocytes (arrowheads). Cells in in all specimens by incubations with non-immune rabbit hypotonic buffer are outlined for ease of size comparison. (B and C) serum and without primary antibody. Time course of osmotic swelling of cholangiocytes. Cells were exposed to either 300 mOsm (o), 200 mOsm (o), 100 mOsm (o), or RESULTS 30 mOsm (A) buffer. (B) Cholangiocyte size assessed by quantitative phase-contrast microscopy. Results reflect measurements of
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