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(Received for publication, February 24, 1995; and in revised form, January
3, 1996) From the
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
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 through the lipid portion of the bilayer or through discrete
membrane proteins that form nonselective (e.g. glucose
transporters) or selective water channels(5) . Recently, the
aquaporins, a family of intrinsic membrane proteins that function as
water-selective channels, were identified and their members localized
in the plasma membranes of cells of many water-transporting
tissues(6) . Aquaporin-CHIP (CHannel-forming Integral Protein of 28 kDa) ( Recently, 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.
Calcein was selected for the following reasons: (a) calcein
labels exclusively cytosol (16) and, during hypotonic swelling,
hepatocyte cytosol shows identical changes to those of the whole cell (19) ; (b) calcein emits pH insensitive fluorescence; (c) calcein causes no effect on osmotic hepatocyte swelling as
judged by quantitative phase-contrast microscopy of hepatocytes with
and without calcein loading (data not shown); and (d)
spontaneous and swelling-induced calcein leakage from hepatocytes
during the time of the experiments was negligible (less than 2%). The P
Membrane fluidity was estimated by measuring steady-state anisotropy
by fluorescence polarization as described previously(28) .
Briefly, 100 µg of membrane protein were added to 2.0 ml of 250
mM sucrose buffer containing 5 µl of 1 mM 1,6-diphenyl-1,3,5-hexatriene (Molecular Probes) and allowed to
equilibrate for 1 h. Steady-state anisotropy was measured in an SLM
4800 spectrofluorometer (SLM, Urbana, IL) with polarization filters
parallel and perpendicular to the excitation beam. Measurements were
made at 25 °C using an excitation wavelength of 362 nm and emission
wavelength of 420 nm.
Figure 1:
Osmotic water movement by rat
hepatocytes. A, phase-contrast micrographs of rat hepatocytes
in isotonic (300 mosM) (top panels) and hypotonic (30
mosM) (bottom panels) buffers at 23 °C. Note the
immunomagnetic beads (arrowheads) around hepatocytes as the
internal standards. Magnification,
Hepatocyte volume changes in hypotonic Hepes-buffered saline buffer
(100 mosM) was also analyzed by laser scanning confocal
microscopy followed by three dimensional reconstruction. Relative cell
volume values obtained using this methology (155.2 ± 14.0, n = 7) agree with the corresponding values shown in Fig. 1B using quantitative phase-contrast microscopy.
Importantly, the viability of hepatocytes (as assessed by trypan blue
exclusion) was unchanged after exposure to hypotonic buffers. Using
the initial slope of the curves generated in Fig. 1B,
the calculated P
Figure 2:
Effect of temperature on osmotic-induced
hepatocyte swelling. A, time course of osmotic-induced
hepatocyte swelling at different temperatures. Cells were exposed to 30
mosM buffer at either 10 °C (
Preincubation of
hepatocytes with amphotericin B significantly (p < 0.0001)
increased both the rapidity and the magnitude of the increase in
hepatocyte volume following exposure to hypotonic (100 mosM)
buffer (Fig. 3A) at 10 °C. Moreover, the effect of
buffer temperature on osmotic-induced water movement by hepatocytes was
significantly reduced following pretreatment with amphotericin B (Fig. 3B). Accordingly, the E
Figure 3:
Effect of amphotericin B on water movement
across the plasma membrane of hepatocytes. A, effect of
amphotericin B on the time course of osmotic-induced hepatocyte
swelling. Cells were exposed to 100 mosM buffer in the absence of
amphotericin B (
Figure 4:
Gene expression of water channels in rat
hepatocytes. Gel electrophoresis of products obtained by RT-PCR using
specific oligonucleotides to the rat MIWC gene (A) and AQP-CD (B). Lane 1, molecular weight markers; lane
2, water (negative control); lane 3, rat kidney (positive
control); and lane 4, rat hepatocytes. For each reaction, 1
µg of total RNA was used as template, except the negative control,
water.
The major findings of the study relate first to functional
and molecular studies of water transport in isolated hepatocytes and
second to a comparative analysis of the membrane lipid compositions of
rat hepatocytes and cholangiocytes, two hepatic epithelia which
transport water via different mechanisms. 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) . We observed no hepatocyte volume
regulation over the time of our experiments. This finding agrees with
recent reports showing that hepatocytes exposed to hypotonic stress
display no significant volume regulatory decrease over 1
min(18, 29) . However, since the changes in cell
volume at 1 min of exposure to hypotonic solutions were smaller than
might be expected for an osmometric cell response, the possibility of a
very rapid cell volume regulation response during the swelling phase
cannot be completely excluded. Nevertheless, since such responses are
likely to have a finite threshold, their contribution to the initial
change in cell volume (from which P Having generated biophysical data consistent with
the notion that the principal mechanism regulating osmotic water
movement by hepatocytes is diffusion via the lipid bilayer, we next
explored this concept at a molecular level by determining whether
hepatocytes express the transcript for three of the previously
described water channels. Using RT-PCR and oligonucleotides based on
reported DNA sequences, we demonstrated that hepatocytes do not express
the transcript for any of the three known water channels,
aquaporin-CHIP, AQP-CD, and MIWC. Thus, both biophysical and molecular
data strongly suggest that the principal mechanism of water movement by
hepatocytes is not channel-mediated, but rather is diffusional. As
recently described by us(13) , cholangiocytes transport water
via a water channel, that is likely, aquaporin-CHIP. In spite of the
presence of water channels, cholangiocytes have a relatively low P As predicted, we found the membrane
lipid composition of hepatocytes to be markedly different from that of
cholangiocytes. Both the cholesterol content and the
cholesterol/phosphoplipid ratio of hepatocyte plasma membranes were
significantly less than those of cholangiocyte membranes. This
difference reflects the unusually high cholesterol content of
cholangiocyte plasma membranes, an observation previously made by us on
plasma membranes derived from cholangiocytes after bile duct
ligation(25) . As expected from the differences in lipid
composition, the membrane fluidity of hepatocytes, estimated by
measuring steady-state anisotropy, was higher than that of
cholangiocytes. Membrane fluidity is recognized to influence
transmembrane transport processes, including water
movement(32) . Thus, these differences in membrane lipid
composition and fluidity between hepatocytes and cholangiocytes may
help to provide a biophysical explanation for their different
mechanisms of water transport; i.e. water can easily diffuse
across the highly fluid hepatocyte plasma membrane but requires a
channel to traverse the stiff cholangiocyte plasma membrane. Historically, the paracellular pathway has been considered by some
to be the principal route of water movement across hepatocytes during
primary bile formation, involving passive movement of water from blood
to bile between hepatocytes in response to osmotic gradients
established largely by the active movement of bile acids into the
canaliculus. This concept stems from inferences made from
ultrastructural studies of hepatocytes demonstrating: (i) enhanced
tight junction penetration of electron-dense substances under
choleretic conditions (33, 34) and (ii) balloon-like
projections in the basolateral (sinusoidal) membrane adjacent to tight
junctions in association with bile acid-stimulated
choleresis(34) . Furthermore, the demonstration that hepatocyte
couplets have low electrical resistance (35) is consistent
with hepatocytes being ``leaky'' epithelium through which
paracellular movement of water may occur. Nevertheless, while current
opinion appears to favor a paracellular pathway of water movement
across hepatocytes, the studies on which this premise is based are
limited and largely indirect. Although our data suggest that
osmotic-induced transmembrane water movement by hepatocytes does not
occur via a channel-mediated mechanism, the P
Volume 271,
Number 12,
Issue of March 22, 1996 pp. 6702-6707
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
10 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).
)was the
first water channel identified(7) ; it has been characterized
biophysically, expressed in Xenopus oocytes(8) , and
reconstituted into proteoliposomes (9) . Two other water
channels have subsequently been isolated by screening rat cDNA
libraries for homology with aquaporin-CHIP(10, 11) :
the water channel of the collecting duct (AQP-CD) and a
mercurial-insensitive water channel (MIWC). Both the AQP-CD and the
MIWC have similar biophysical properties to aquaporin-CHIP, including a
high osmotic water permeability coefficient (P) and low activation energy (E
), the latter reflecting temperature
independence of water movement, an important criterion for
channel-mediated water transport(5, 12) . As with
aquaporin-CHIP, osmotic-induced water movement through the AQP-CD is
inhibited by mercurial compounds. Water movement through the MIWC lacks
mercury sensitivity because of a substitution of the mercury-sensitive
cysteine residue with alanine(11) .
Preparation of Hepatic Epithelial Cells
Hepatic epithelial cells were isolated from male Fisher 344
rats (250-350 g) obtained from Harlan Sprague-Dawley, Inc.
(Indianapolis, IN). Rats were provided with standard laboratory diet
and water ad libitum and anesthetized with pentobarbital (50
mg/kg, intraperitoneal).Hepatocytes
Pure (>98%) preparations of hepatocytes
were isolated from rat livers by collagenase perfusion, mechanical
dissociation, and isopycnic centrifugation through a discontinuous
Percoll gradient as described previously(14) .Cholangiocytes
Pure (>95%) preparations of
cholangiocytes were isolated from rat livers using an immunoaffinity
technique previously described by us(15) . Cell viability as
assessed by trypan blue exclusion was always greater than 90% for both
cell preparations.Osmotic Water Permeability Studies
Cell Volume Measurements
The size of cells in
isotonic and hypotonic extracellular buffers was assessed by
quantitative phase contrast microscopy as described previously by
us(13) . Briefly, freshly isolated cells were attached to 22-mm
square glass coverslips with Cell tak (Collaborative Biomedical
Products, Bedford, MA) and mounted on the stage of an Olympus CK21
inverted phase contrast microscope (Leeds Precision Instruments,
Minneapolis, MN) equipped with an Olympus SC35 type 12 camera. Cells
were bathed in isotonic (300 mosM) Hepes-buffered saline
containing (in mM): 140 NaCl; 5.4 KCl; 0.8
Na
HPO
; 25 Na-Hepes; 0.8 MgSO; pH
7.4, 23 °C. Studies were performed by exposing cells at time 0 to
extracellular buffers of different osmolarity (range: 30-300
mosM); buffers were prepared by diluting Hepes-buffered saline
with distilled water and measuring osmolarity with an Osmette S
osmometer (Precision Instruments Inc., Natick, MA). Serial slide
photographs were taken at 400 magnification and cell diameters
measured in a randomized, blinded manner from projected images using
the 4.5-µm immunomagnetic beads as internal standards for cell size
measurements. Cell volumes and surface areas were calculated from cell
diameter measurements using the fact that freshly isolated hepatocytes
and cholangiocytes are spherical in shape(14, 15) ,
and results are expressed as percent change in cell volume over time.
Relative cell volume values obtained by quantitative phase-contrast
microscopy were validated by two independent methods: (a)
laser scanning confocal microscopy of calcein-loaded hepatocytes (16) and computer-assisted three-dimensional reconstruction and
volume calculation; and (b) sequential phase-contrast and
fluorescence microscopy (17) of hepatocytes loaded with calcein
and computer-assisted measurements of the corresponding diameters and
volumes. Additionally, we tested a possible rapid volume regulatory
decrease during the time of our experiments using iso- and hypotonic
Hepes (25 mM)-buffered sucrose(18) .Laser Scanning Confocal Microscopy
Cell images
were collected with an inverted Zeiss laser scanning confocal
microscope. Serial confocal fluorescence images of calcein-loaded
hepatocytes were collected as the focal plane was advanced in 1-µm
increments through the cell thickness(16) . Images from 40
focal planes were collected (total time of the process approximately 1
min). Fluorescence excitation was 488 nm and the emission collected
above 520 nm. Confocal images were transferred to a Silicon Graphics
Workstation operating ANALYZE software (Mayo Foundation)
for three-dimensional reconstruction and volume calculation.
Sequential Phase-contrast and Fluorescence
Microscopy
Images of calcein-loaded hepatocytes were taken on an
Attofluor RatioVision System (Atto Instruments, Inc., Rockville, MD) (17) . The computer-controlled filter changer selects between
phase-contrast and fluorescence. A Zeiss Axiovert 135 TV inverted
microscope was equipped with a 40 Phase2 LD Achroplan objective
lens and an intensified CCD camera. Fluorescence excitation was 488 nm,
and the emission collected above 520 nm.Dye Loading
Calcein was loaded by incubating
hepatocytes in culture medium with 7 µmol/liter calcein-AM
(Molecular Probes, Eugene, OR) for 30 min at 37 °C. Cell viability
was simultaneously checked with ethidium homodimer (Molecular Probes)
which is excluded by the intact plasma membranes of living cells. 
(cm/s) of hepatocytes was calculated from
osmotic swelling data, initial hepatocyte volume (V
= 9.93 10 cm
) and
surface area (S = 22.95 10 cm
), and the molar volume of water (V
= 18 cm/mol), as described
elsewhere(13, 20) . Experiments were performed at
different temperatures (range: 10-30 °C); the E
was derived from the Arrhenius relation between P values and temperature as
described(13, 21) . In select experiments, the osmotic
water permeability of hepatocytes was measured in the presence of
amphotericin B (300 µg/ml) to determine whether the insertion of
artificial pores into the hepatocyte plasma membrane would
significantly alter the biophysical properties of transmembrane water
movement.Water Channel Gene Expression in Hepatic Epithelia
Total cellular RNA was isolated from whole organs and pure
preparations of hepatocytes and cholangiocytes as described previously (13, 22) . Specific oligonucleotide DNA primers were
prepared based on the published sequences of the three previously
described water channels, aquaporin-CHIP, AQP-CD, and
MIWC(10, 11, 23) . Using these primers and
total cellular RNA as template, complementary DNA sequences were
generated using reverse transcriptase-polymerase chain reaction
(RT-PCR). These PCR products were identified by gel electrophoresis,
sequenced as described previously(24) , and compared to the
published sequence for each of these water
channels(10, 11, 23) .Membrane Lipid Composition and Fluidity of Hepatic
Epithelia
Membrane vesicles were prepared from purified cholangiocyte
and hepatocyte plasma membranes as described previously by us (25) and others(26, 27) . Total membrane
cholesterol was measured spectrophotometrically using a commercially
available kit (Boehringer Mannheim). Total membrane phospholipid was
measured with a commercially available kit (Wako Chemicals U. S. A.
Inc., Richmond, VA) using the protocol supplied by the manufacturer.
Osmotic Water Permeability Studies
As shown in Fig. 1, hepatocytes rapidly and significantly increased in cell
size within the first 60 s of exposure to hypotonic buffers. 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). The magnitude of the increase
in hepatocyte volume in hypotonic buffers was time-dependent (p < 0.0001, analysis of variance) in contrast, cells exposed to
300 mosM isotonic buffer remained the same size over time (Fig. 1, A and B). These results were
confirmed by sequential phase-contrast and fluorescence microscopy of
calcein-loaded hepatocytes exposed to hypotonic Hepes-buffered saline
or sucrose buffers for up to 1 min (Table 1). These data indicate
that substitution with sucrose of permeant ions involved in volume
regulation produced no significant differences in hepatocyte osmotic
response, suggesting that cell volume regulation did not contribute
substantially to the response over the time of our experiments.
400. B, time course
of osmotic-induced hepatocyte swelling assessed by quantitative phase
contrast microscopy. Cells were exposed to either 300 mosM (
), 200 mosM (
), 100 mosM (
),
or 30 mosM () buffer at 23 °C. Results represent
mean ± S.D. from measurement of more than 30 hepatocytes for
each time point.
value for isolated rat
hepatocytes was 66.4 10 cm/s at 23 °C.
The effect of extracellular buffer temperature on the volume of
hepatocytes in hypotonic buffer is shown in Fig. 2A.
Temperature had a significant (p < 0.0001, analysis of
variance) effect on the time-dependent increase in hepatocyte volume in
hypotonic (30 mosM) buffer; the magnitude of the increase in
hepatocyte volume increasing with increasing buffer temperature.
Indeed, when comparing studies done at 23 °C and 10 °C, the
osmotic water permeability coefficient decreased by 55% (p < 0.001 at 23 °C, t test). In contrast, the
magnitude of the time-dependent increase in cholangiocyte volume in
hypotonic (30 mosM) buffer was not significantly different
between studies at 10, 23, or 30 °C (data not shown). From these
data, we determined the Arrhenius relationship between the logarithm of P
and the reciprocal value of absolute temperature
for both hepatocytes and cholangiocytes as shown in Fig. 2B. Based on these data, the E value for hepatocytes was 12.8 kcal/mol.
), 23 °C (),
or 30 °C (). Results represent mean ± S.D. from
measurements of more than 30 hepatocytes. B, the Arrhenius
relation between the logarithm of P and
reciprocal of absolute temperature. These values for both hepatocytes
(
) and cholangiocytes () had a linear relationship. The E values were calculated from those
slopes. Results represent mean ± S.D. from measurements of more
than 30 hepatocytes.
value for hepatocytes in the presence of amphotericin B was 3.6
kcal/mol, a value similar to cholangiocytes and compatible with
channel-mediated water movement. Of note, the viability of hepatocytes
in the presence of amphotericin B was > 90% indicating that the
stimulatory effect of amphotericin B on hepatocyte water movement was
not due to cell toxicity.
) or to 100 mosM buffer containing 300
µg/ml amphotericin B after 10-min preincubation with 300 µg/ml
amphotericin B () at 10 °C. Results represent mean ±
S.D. from measurements of more than 30 hepatocytes. B, effect
of temperature on P values for
hepatocytes in the presence and absence of amphotericin B. The P
values were calculated from the data
generated from measurements of hepatocyte swelling in 100 mosM buffer in the absence of amphotericin B (white bar) or in
the presence of 300 µg/ml amphotericin B (black bar) at
either 10, 23, or 30 °C. Results represent mean ± S.E. from
measurements of more than 30 hepatocytes.
Gene Expression of Water Channels
Shown in Fig. 4A are the results from a gel electrophoresis of
products obtained by RT-PCR using specific oligonucleotides for rat
MIWC. The gel shows a band at 500 bp in the lane where RNA isolated
from rat kidney was used as a template, our positive control. No band,
however, was detected in the lane where an equal amount of RNA from
purified rat hepatocytes was used as template, nor in the lane
containing water as template, our negative control. By DNA sequencing,
the band obtained using rat kidney RNA was greater than 95% homologous
to the rat brain MIWC sequence(11) . Similarly, Fig. 4B shows the results from a gel electrophoresis of products obtained
by RT-PCR using specific DNA primers for rat AQP-CD. As expected, the
gel shows a band at 504 bp in the lane where RNA isolated from rat
kidney was used as template, our positive control; by DNA sequencing,
the band obtained using rat kidney RNA was greater than 95% homologous
to the rat kidney AQP-CD sequence. Note however, that no band was
detected using an equal amount of RNA from purified rat hepatocytes as
template, nor when using water, our negative control. Finally, as
previously reported by us(13) , no band was detected on gel
electrophoresis of products obtained by RT-PCR using specific primers
for aquaporin-CHIP and hepatocyte total RNA as template(13) .
Membrane Lipid Composition and Fluidity
Table 2shows the results of membrane lipid analysis and
steady-state anisotropy for hepatocytes and cholangiocytes. The
cholesterol content and cholesterol/phospholipid ratio of hepatocyte
membranes were both significantly (p < 0.005) less than
those of cholangiocyte membranes. In particular, the
cholesterol/phospholipid ratio of hepatocyte membranes was
approximately 8-fold less than that of cholangiocytes. It is well
documented that the cholesterol/phospholipid ratio is one of the
important parameters affecting membrane fluidity. Thus, as expected,
steady-state anisotropy measurements of hepatocyte membranes yielded
significantly (p < 0.005) lower values than that obtained
from cholangiocyte membranes.
was estimated)
would likely be small. Several lines of evidence indicate that P was not substantially restricted by non-membrane
barriers, such as external or cytoplasmic unstirred layers (30) : (a) there was no lag in hepatocyte swelling
after a change in buffer osmolarity; (b) E was 12.8 kcal/mol, much higher than that predicted if P were limited by an unstirred layer (5
kcal/mol); (c) P
increased with insertion
of amphotericin B water channels; and (d) the initial rate of
cell swelling was proportional to osmotic gradient size (data not
shown). The calculated P and E values, together with the fact that amphotericin B-induced
membrane channels increased P and lowered E, are consistent with water movement through the
lipid portion of the hepatocyte plasma membrane rather than through
protein channels. value of 50 10 cm/s.
Although our data suggest that water movement by hepatocytes is due to
diffusion via lipid bilayer, the P
value for
hepatocytes is higher than that of cholangiocytes. Furthermore,
cholangiocytes have a markedly lower diffusional water permeability
coefficient (P
), <5 10 cm/s, compared to that of hepatocytes(31) . Indeed, to
our knowledge, this P
value for cholangiocytes
represents the lowest P value reported thus
far(5) . These important differences in the mechanisms by which
water traverses hepatocytes and cholangiocytes suggested that there
might be major differences in the biochemical properties of the plasma
membranes of these two epithelia. Nevertheless, results of such
comparisons must be interpreted with caution because both P and P values are dependent
on cell surface area, and these two cell types could differ with
respect to their surface areas. value for hepatocytes is higher than the value for cholangiocytes
which have aquaporin-CHIP water channels in their plasma membranes.
Thus, it seems plausible that diffusional water movement across
hepatocytes in response to osmotic gradients may play an important role
in primary bile formation at bile canaliculi. Currently, however, this
notion is speculative since the relevance of our in vitro data
to the in vivo situation is unclear. Indeed, we obtained P values in isolated hepatocytes which rapidly
lose their polarity; thus, these values do not necessarily reflect the
osmotic permeability of the canalicular membrane. Of course, this
concept does not exclude a paracellular pathway in water movement
across hepatocytes nor does it exclude the possibility of other
nonselective, membrane channels such as glucose transporters from
playing a contributory role in water movement(36) .
Unfortunately, it is currently difficult to directly distinguish
transcellular from paracellular water movement in vivo,
because of the lack of a suitable experimental model. Additional
studies requiring new experimental approaches will be required to
determine the quantitative contribution of a transcellular versus a paracellular pathway in primary bile formation by hepatocytes.
Nevertheless, the work described here excludes selective water channels
as important conduits for transcellular water movement across
hepatocyte plasma membranes. Moreover, the biophysical properties of
hepatocyte plasma membranes characterized by us clearly indicate that a
diffusional mechanism for transcellular water movement could be very
important in primary bile formation.
), osmotic water permeability
coefficient; P
, diffusional water
permeability coefficient; E
, activation
energy; RT, reverse transcriptase; PCR, polymerase chain reaction.
We thank Dr. D. Braddock for his valuable help in
confocal microscopy and Maureen Craft for typing the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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Y. Koyama, T. Yamamoto, D. Kondo, H. Funaki, E. Yaoita, K. Kawasaki, N. Sato, K. Hatakeyama, and I. Kihara Molecular Cloning of a New Aquaporin from Rat Pancreas and Liver J. Biol. Chem., November 28, 1997; 272(48): 30329 - 30333. [Abstract] [Full Text] [PDF]
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R. A. Marinelli, L. Pham, P. Agre, and N. F. LaRusso Secretin Promotes Osmotic Water Transport in Rat Cholangiocytes by Increasing Aquaporin-1 Water Channels in Plasma Membrane. EVIDENCE FOR A SECRETIN-INDUCED VESICULAR TRANSLOCATION OF AQUAPORIN-1 J. Biol. Chem., May 16, 1997; 272(20): 12984 - 12988. [Abstract] [Full Text] [PDF]
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F. Garcia, A. Kierbel, M. C. Larocca, S. A. Gradilone, P. Splinter, N. F. LaRusso, and R. A. Marinelli The Water Channel Aquaporin-8 Is Mainly Intracellular in Rat Hepatocytes, and Its Plasma Membrane Insertion Is Stimulated by Cyclic AMP J. Biol. Chem., April 6, 2001; 276(15): 12147 - 12152. [Abstract] [Full Text] [PDF]
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M. W. Roe, A. L. Moore, and S. D. Lidofsky Purinergic-independent Calcium Signaling Mediates Recovery from Hepatocellular Swelling. IMPLICATIONS FOR VOLUME REGULATION J. Biol. Chem., August 10, 2001; 276(33): 30871 - 30877. [Abstract] [Full Text] [PDF]
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