Am J Physiol Regul Integr Comp Physiol 289: R877–R890, 2005.
First published May 19, 2005; doi:10.1152/ajpregu.00170.2005.
Importance of cytoskeletal elements in volume regulatory responses
of trout hepatocytes
Hannes L. Ebner,1,2 Alexandra Cordas,1 Diego E. Pafundo,3 Pablo J. Schwarzbaum,3
Bernd Pelster,1,2 and Gerhard Krumschnabel1,2
1
Institut für Zoologie und Limnologie, and 2Center of Molecular Biosciences, Leopold Franzens
Universität Innsbruck, Innsbruck, Austria; and 3Instituto de Quı́mica y Fisicoquı́mica Biológicas
(Facultad de Farmacia y Bioquı́mica), Universidad de Buenos Aires, Buenos Aires, Argentina
Submitted 9 March 2005; accepted in final form 17 May 2005
Oncorhynchus mykiss; F-actin; microtubule; acid secretion; intracellular pH; intracellular sodium; intracellular free calcium
is of pivotal importance for the
maintenance of structural integrity, a constant intracellular
milieu, and, ultimately, cell survival. Thus, after volume
changes evoked by anisotonic conditions, most cells are capable of restoring their cell volume by activating various mechanisms, mediating a net loss or uptake of osmolytes as required
(2). Particularly in mammalian cells, the various transporters
involved in these volume regulatory responses have been well
characterized over the past decades (64), but there is still a
considerable gap in our knowledge of the factors controlling
THE REGULATION OF CELL VOLUME
Address for reprint requests and other correspondence: G. Krumschnabel,
Institut für Zoologie und Limnologie, Leopold Franzens Universität Innsbruck,
Technikerstrasse 25, A-6020 Innsbruck, Austria (e-mail: gerhard.krumschnabel
@uibk.ac.at).
http://www.ajpregu.org
and regulating their activity. One aspect that has received
increasing interest during the last years is the role of cytoskeletal elements, including microfilaments, microtubules, and associated proteins (53). Concerning the actin-based cytoskeleton, several studies have shown that anisotonic changes of cell
volume are associated with significant changes of cellular
F-actin content and/or distribution (29, 50, 54, 66). Conversely,
disruption of F-actin has been shown to delay or even totally
inhibit volume regulation in a number of cell models (19, 44,
54). Similar results have been reported with regard to the
microtubule network, although this has been less thoroughly
studied (15, 19, 28). The mechanistic basis of these effects is
not yet fully understood, but various reports suggest that
cytoskeleton elements may directly control the activity of a
number of transporters (40, 43, 48) or participate in the process
of vesicle-mediated insertion and retrieval of transport proteins
(53).
Based on these results, the importance of the cytoskeleton as
a factor involved in volume regulatory responses is widely
accepted, but there also exist a number of studies in which
cytoskeletal disrupting agents had no effect on volume regulation (12, 25, 42) or in which anisotonic volume changes did
not elicit quantitative changes in the abundance of cytoskeletal
elements (29). Although these differences may be partly attributed to differences in the experimental approaches applied,
there also is some evidence that they may reflect genuine cell
type-specific differences, at least for mammalian cells. With
regard to nonmammalian cells, in addition to older studies on
frog gallbladder (19), shark rectal gland (35, 66), and vesicles
from Necturus enterocytes (16), there also is a recent report on
eel intestinal cells (45) demonstrating a close link between the
cytoskeleton and volume regulation. All of these cell types
studied are specialized epithelial cells exposed to severe anisotonic challenges on a regular basis. However, to our knowledge, there is no study yet available on isolated hepatocytes
from lower vertebrates, such as rainbow trout hepatocytes,
which may be taken as a representative model for epithelial
cells that are located in a more constant osmotic environment
because they are not facing the environmental water. Furthermore, the trout hepatocyte is a well-established model system
for the study of lower vertebrate cell function, for which
several studies have investigated the capability of volume
recovery under hypo- and hypertonic conditions and the transport mechanisms involved in volume regulation (5, 6, 20, 37,
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6119/05 $8.00 Copyright © 2005 the American Physiological Society
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Ebner, Hannes L., Alexandra Cordas, Diego E. Pafundo, Pablo
J. Schwarzbaum, Bernd Pelster, and Gerhard Krumschnabel. Importance of cytoskeletal elements in volume regulatory responses of trout
hepatocytes. Am J Physiol Regul Integr Comp Physiol 289: R877–R890,
2005. First published May 19, 2005; doi:10.1152/ajpregu.00170.2005.—
The role of cytoskeletal elements in volume regulation was studied in
trout hepatocytes by investigating changes in F-actin distribution
during anisotonic exposure and assessing the impact of cytoskeleton
disruption on volume regulatory responses. Hypotonic challenge
caused a significant decrease in the ratio of cortical to cytoplasmic
F-actin, whereas this ratio was unaffected in hypertonic saline. Disruption of microfilaments with cytochalasin B (CB) or cytochalasin D
significantly slowed volume recovery following hypo- and hypertonic
exposure in both attached and suspended cells. The decrease of net
proton release and the intracellular acidification elicited by hypotonicity were unaltered by CB, whereas the increase of proton release in
hypertonic saline was dramatically reduced. Because amiloride almost
completely blocked the hypertonic increase of proton release and
cytoskeleton disruption diminished the associated increase of intracellular pH (pHi), we suggest that F-actin disruption affected Na⫹/H⫹
exchanger activity. In line with this, pHi recovery after an ammonium
prepulse was significantly inhibited in CB-treated cells. The increase
of cytosolic Na⫹ under hypertonic conditions was not diminished but,
rather, enhanced by F-actin disruption, presumably due to inhibited
Na⫹-K⫹-ATPase activity and stimulated Na⫹ channel activity. The
elevation of cytosolic Ca2⫹ in hypertonic medium was significantly
reduced by CB. Altogether, our results indicate that the F-actin
network is of crucial importance in the cellular responses to anisotonic
conditions, possibly via interaction with the activity of ion transporters and with signalling cascades responsible for their activation.
Disruption of microtubules with colchicine had no effect on any of the
parameters investigated.
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CYTOSKELETON AND VOLUME REGULATION IN HEPATOCYTES
MATERIALS AND METHODS
Materials. Collagenase (type VIII), bovine serum albumin (BSA),
fetal calf serum (FCS), ionophores, antibiotics, CB, CD, colchicine,
amiloride, 4-acetamido-4⬘-isothiocyanostilbene-2,2⬘-disulfonic acid
(SITS), latrunculin B, ouabain, and poly-L-lysine all were purchased
from Sigma (Deisenhofen, Germany). The acetoxymethyl esters (AM)
of 2⬘,7⬘-bis-(2-carboxypropyl)-5(6)-carboxyflourescein (BCPCF),
fura-2, calcein, and sodium-binding benzofuran isophthalate (SBFI)
and Alexa Fluor phalloidin were from Molecular Probes (Leiden,
Netherlands). Cariporide was a kind gift from Sanofi-Aventis Pharma
(Vienna, Austria). Leibovitz L-15 medium was bought from Invitrogen. The mouse anti ␤-tubulin monoclonal antibody (MAb 3408) was
obtained from Chemicon, the TRITC-labeled anti mouse secondary
antibody was from DAKO, and Vectashield mounting medium was
from Vector Laboratories. All other chemicals were of analytical
grade and were purchased from local suppliers.
Preparation of hepatocytes and cell culture. Rainbow trout, Oncorhynchus mykiss, were obtained from a local hatchery. Animals
were acclimated at 15°C in 200-liter aquaria and were daily fed ad
libitum with trout pellets. Trout were killed with a sharp blow on the
head and subsequent transection of the spinal cord. Maintenance and
use of experimental animals were in accordance with the Austrian
federal law for care and use of laboratory animals. Hepatocytes were
isolated using a method based on collagenase digestion and differential centrifugation, as described previously (36). After cell isolation,
hepatocytes were suspended in standard saline (for composition,
Experimental media) with 1% BSA and were left to recover for 1 h in
a shaking water bath thermostated to 19°C, which also was the
temperature used during the experiments. Cells were then counted,
and viability was determined using the Trypan blue exclusion method.
For the determination of cell volume, acid secretion, and Rb⫹ uptake,
these freshly isolated cells were used. Intracellular ion concentrations
(H⫹, Na⫹, Ca2⫹) and cytoskeleton elements were assessed in hepatocytes attached to glass coverslips. For this purpose, cells were
diluted to a concentration of 2 ⫻ 106 cells/ml in modified Leibovitz
L-15 medium containing 10% FCS, 10 mM HEPES, 5 mM NaHCO3,
50 ␮g/ml gentamicin, and 100 ␮g/ml kanamycin, titrated to a final pH
of 7.6. Hepatocytes were plated onto poly-L-lysine-pretreated glass
coverslips and left to adhere overnight in an incubator at 19°C and
0.5% CO2. Cells were washed three times with standard saline to
remove damaged cells and debris before further use.
Experimental media. The standard incubation medium with an
osmolarity of 284 mosM consisted of (in mM) 10 HEPES, 136.9,
NaCl, 5.4 KCl, 1 MgSO4, 0.33 NaH2PO4, 0.44 KH2PO4, 1.5
AJP-Regul Integr Comp Physiol • VOL
NaHCO3, 1.5 CaCl2, and 5 glucose, pH 7.6, at 19°C. Hypotonic
medium with an osmolarity of 166 mosM consisted of a mixture of
equal volumes of standard saline and the same medium without NaCl
(0.58⫻ isosmolarity). Similarly, a mixture of standard saline and
saline containing an additional 200 mM NaCl was used to create
hypertonic conditions, yielding an osmolarity of 465 mosM (1.6⫻
isosmolarity). Medium osmolarity was assessed by freezing point
depression using a Knaus Semi-Micro osmometer (Berlin, Germany).
Experimental exposure, fixation, and staining of hepatocytes for
confocal microscopy. Primary cell cultures were carefully washed
with standard saline and then incubated in this medium for at least 1 h.
Subsequently, cells were exposed to one of the following treatments.
1) Hepatocytes were incubated with 2 ␮M CB in standard saline for
30 min or, for comparative reasons, with 2 ␮M of the analog CD for
15 min, or with 2 ␮M latrunculin B for 15 min. The latter compound
leads to a disruption of F-actin by a mechanism differing from that of
the cytochalasins. As established in preliminary studies and shown
below, these concentrations of CB, CD, and latrunculin B were found
suitable to cause a visible disruption of the F-actin network. 2) Cells
were incubated with 100 ␮M colchicine in isotonic medium for 30
min, which produced a decrease of visible microtubule elements. 3)
Cells were exposed to hypotonic conditions for 5 and 30 min. 4)
Hepatocytes were incubated in hypertonic saline for 5 and 30 min. At
the end of the respective incubation period, hepatocytes were washed
twice with phosphate-buffered saline (PBS, pH 7.6) and fixed for 60
min at room temperature with 4% paraformaldehyde in PBS. For
confocal visualization of actin and tubulin, cells were permeabilized
by treatment with PBS containing 0.1% Triton X-100 and then
blocked over 1 h in PBS containing 0.1% Triton X-100, 1% BSA, and
10% FCS. Subsequently, cells were incubated overnight in a humidified dark chamber at ⫹4°C with a 1:200 dilution of mouse anti
␤-tubulin monoclonal antibody and a 1:150 dilution of Alexa Fluor
phalloidin. After three washes in PBS with 0.1% Triton X, the
coverslips were further incubated in the dark chamber for 1 h in the
presence of TRITC-labeled anti mouse secondary antibody (1:150) at
room temperature, and finally, after additional washings, the slides
were covered with mounting medium and fixed on object slides. For
the analysis of actin only, the overnight incubation step, as well as
incubation with secondary antibody, was omitted. Cells were visualized with a confocal laser scanning fluorescence microscope (LSM
510; Zeiss) with excitation and emission wavelengths of 488 and
505–550 nm for Alexa Fluor phalloidin and of 543 and ⬎585 nm for
TRITC. Three-dimensional reconstruction images of the hepatocytes
were created from serial 1-␮m optical slices acquired using the LSM
510 software (version 3.2; Zeiss).
F-actin quantification. Quantification of relative F-actin distribution was made using confocal laser scanning microscopy by applying
the method described by Erickson et al. (17). This method is based on
the observation that, similar to chondrocytes (17), F-actin of resting
hepatocytes is predominantly localized near the plasma membrane
and that it is this cortical actin that is mainly affected by cell swelling
or shrinkage. Accordingly, the ratio of cortical actin fluorescence to
that of cytoplasmic actin proved to be a sensitive measure to quantify
changes in the actin-cytoskeleton. By determining the ratio, this
method also circumvents the problem of variations in staining efficiency between individual cell cultures. For these measurements,
F-actin was labeled with the fluorescent phalloidin derivative in fixed
cells as described above. Images for these measurements were captured using a plan Neofluar ⫻40/1.30 oil objective lens with a large
pinhole setting resulting in an optical slice thickness of 7 ␮m. With
the use of Zeiss software, a linear intensity profile was laid through the
equatorial plane of individual cells, and the average of the peak
intensities measured near the cell membrane (Imembrane) and that of
the cytoplasmic region between these two points was determined
(Icytoplasm). F-actin distribution was then calculated according to the
following formula: actin ratio ⫽ (Imembrane ⫺ Icytoplasm)/(Imembrane ⫹
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49, 52). However, information concerning the interaction between cytoskeleton and volume regulation of these cells is
missing so far. It is therefore not known whether trout hepatocytes behave similarly to their mammalian counterparts in
this regard or if they display deviating response patterns, as
recently has been observed with respect to the metabolic
alterations elicited by anisotonic conditions (37).
In the present study we therefore aimed at addressing the
role of cytoskeletal elements for volume regulation in trout
hepatocytes. The goals of our study were to evaluate 1)
alterations of cytoskeletal elements, specifically of the actinbased cytoskeleton, during anisotonic exposure of the hepatocytes; 2) the impact of cytochalasin B (CB) and D (CD),
F-actin disrupting agents, on volume regulation in anisotonic
conditions; and 3) the effect of cytoskeletal disruption on ion
movements associated with volume regulatory responses. In
addition, we studied the effect of the microtubule disrupting
agent colchicine on some parameters of interest to evaluate the
possible importance of microtubules in volume regulation.
CYTOSKELETON AND VOLUME REGULATION IN HEPATOCYTES
AJP-Regul Integr Comp Physiol • VOL
saline or medium containing 2 ␮M CB, 2 ␮M CD, or 100 ␮M
colchicine. After another 15 (CD) or 30 min (CB, colchicine) of
superfusion, the saline was switched again to either hypo- or hypertonic saline, and proton release was determined for an additional 30
min. For cells treated with CB, CD, or colchicine, these agents were
included in the anisotonic saline. To account for possible effects of
medium osmolarity on the sensor signal, we conducted preliminary
experiments in the absence of cells, showing no effect of hypotonic
medium and a very minor increase of the signal in hypertonic saline.
The latter amounted to ⬍5% of the cell signal and was subtracted
from the signals measured with hepatocytes.
Intracellular pH. Intracellular pH (pHi) of attached individual
hepatocytes was measured using the pH-sensitive fluorescent dye
BCPCF, as described previously (38). Cells loaded with the fluorophore were mounted in a stainless steel measuring chamber and fixed
on an inverted fluorescence microscope (Axiovert, Zeiss) equipped
with a ⫻40 UV objective. Fluorescence was determined at 60-s
intervals with excitation at 440 and 490 nm and emission detected
above 510 nm. Hyper- and hypotonic conditions were created by
exchanging 500 ␮l of the standard saline (total volume 1 ml) with
anisotonic medium yielding the desired final osmolarity. Recovery of
pHi from an acid load was determined using the NH⫹
4 prepulse
technique (8, 47). For this purpose, cells were exposed to 30 mM
NH4Cl for 5 min, followed by rapid washing with standard saline. At
the end of each experiment, a three-point calibration was performed
by replacing the experimental media with solutions of defined pH (pH
6.8, 7.2, 7.6) and high K⫹ (medium composition, in mM: 10 HEPES,
5.4 NaCl, 136.9 KCl, 1 MgSO4, 0.33 NaH2PO4, 0.44 KH2PO4, 1.5
NaHCO3, 1.5 CaCl2, and 5 glucose) in the presence of the ionophores
nigericin (10 ␮M) and valinomycin (5 ␮M).
Cytosolic free Na⫹. For the determination of cytosolic free sodium
concentration ([Na⫹]i), cell cultures were incubated for 60 min in the
presence of the fluorescent dye SBFI AM (15 ␮M; 15 mM stock
dissolved in DMSO and 20% Pluronic acid). The loading solution was
then washed out with isotonic medium, and the cells were incubated
for at least another 30 min. Measurements of [Na⫹]i were conducted
on the microscope setup described in Intracellular pH (Zeiss Axiovert) with excitation set to 340 and 380 nm and emission measured
above 510 nm. Calibrations were performed using the method of
Harootunian et al. (27), with minor modifications. In short, the bathing
saline was changed to a Na⫹-free gluconate solution consisting of
136.9 mM K-gluconate, 10 mM HEPES, 5.4 mM KCl, 1 mM MgSO4,
0.77 mM KH2PO4, 5 mM K2HCO3, 1.5 mM CaCl2, and 5 mM
glucose, containing the ionophores gramicidin D (4 ␮M), monensin
(10 ␮M), and nigericin (10 ␮M), with pH 7.6 adjusted with KOH. By
substitution of Na-gluconate for K-gluconate, Na⫹ concentration was
gradually increased from 0 to 20 and 50 mM, and a standard curve
relating the ratio (340/380) to [Na⫹]i was established.
Rb⫹ uptake. Rates of Rb⫹ uptake, serving as a measure of transmembrane K⫹ fluxes, were determined in control cells and in hepatocytes preincubated with 2 ␮M CB for 30 min. Rb⫹ fluxes were
determined both in the presence of 1 mM ouabain, a selective inhibitor
of Na⫹-K⫹-ATPase activity, and in the absence of the inhibitor. The
method used for Rb⫹ uptake measurement has been described in
detail previously (36).
Cytosolic free Ca2⫹. The fluorescent indicator dye fura-2 AM was
used to measure cytosolic free calcium concentration ([Ca2⫹]i), using the
microscope setup described in Intracellular pH (Zeiss Axiovert). Images
of fura-2-loaded cells were captured every 60 s, with settings identical to
those given for SBFI applied. Protocols for anisotonic exposure were the
same as those used for pHi measurements. Fluorescence ratios were
stored, and absolute [Ca2⫹]i were calculated using the formula given by
Grynkiewicz et al. (24), with a dissociation constant (Kd) of 680 nM as
determined previously for our experimental setup using a commercial
calibration kit (Molecular Probes) (37). After each experiment, a maximum fluorescence ratio, obtained by addition of 4.5 mM CaCl2, and a
minimum ratio, obtained after addition of 20 mM EGTA, both in the
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Icytoplasm); these values were normalized to the mean actin ratio
determined in isotonic controls.
Cell water volume. Cell water volume of cultured hepatocytes was
estimated using epifluorescence microscopy according to the method
of Altamirano et al. (1) and modified by Espelt et al. (18). In brief,
coverslips with attached cells were mounted in a chamber filled with
isotonic medium and placed on the stage of a Nikon TE-200 inverted
epifluorescence microscope. Hepatocytes were then loaded with 2 ␮M
calcein AM for 45– 60 min, followed by a period of 1 h during which
the loading solution was washed out with isotonic medium superfused
at a rate of 2 ml/min. Subsequent medium changes were conducted by
hand. Estimates of cell water volume changes were obtained from
changes in fluorescence intensity recorded from a small pinhole
region of each cell as detected through a 500-nm long-pass (LP)
dichroic mirror and a 515-nm LP barrier filter after exciting calcein
though a 470-nm center wavelength excitation filter. Under the conditions applied, changes in calcein fluorescence are proportional to its
concentration and are therefore inversely related to alterations of cell
water volume (18). Calibrations were conducted before each measurement by exposing cells to hypo-, iso-, and hypertonic solutions of
known osmolarity. Osmotic changes induced by the calibration solutions were selected so that no volume regulatory response of the cell
was elicited (18, 52). Data are presented as normalized values of cell
water volume (Vr) as computed from monitored changes in relative
fluorescence.
Volume changes of suspended cells were determined by electronic
cell sizing on a Coulter Counter Multisizer II, with an aperture of 100
␮m. Aliquots of cell suspensions were diluted into 20 ml of iso- or
anisotonic media as given above, and at specified time points,
⬃20,000 cells were measured and their mean volume compared with
that of cells at time t ⫽ 0.
For quantitative comparison of the extent of regulatory volume
changes (RVC) observed in response to anisomotic exposure of the
cells, the initial rate of cell volume recovery (vRVC) and the relative
volume change 20 min (RVC20) or 40 min (RVC40) after maximum
cell swelling or shrinkage (Vrmax and Vrmin, respectively) were
calculated. vRVC was obtained by fitting data of relative volume
change vs. time by the second-order polynomial A0 ⫹ vRVC t ⫹ Ct2,
with the first derivative of this function at time 0 corresponding to
vRVC. RVC20 and RVC40 were calculated as (Vrmax ⫺ Vrt)/(Vrmax ⫺
1) for hypotonic exposure and (Vrmin ⫺ Vrt)/(Vrmin ⫺ 1) for hypertonic exposure, where Vrt corresponds to the relative cell volume at
the respective time after maximum volume change.
Proton secretion. Proton secretion of hepatocytes was assessed
with a cytosensor microphysiometer (Molecular Devices) by measuring the rate of acidification of external medium as described (55), with
modifications (39). To this end, we embedded cells (0.45 ⫻ 106
hepatocytes) in 1.5% low-melting-point agarose on the polycarbonate
membrane of capsule cups and mounted them in the sensor chamber
of the cytosensor microphysiometer. In this chamber, the cells were
superfused via one of two fluid outlets that may be rapidly switched
using an electromagnetic valve. The flow cycle was set to 3 min, with
2.5 min of constant superfusion and a 30-s flow-off period. During
this time, the protons released by the hepatocytes accumulated in the
sensor chamber, and this was registered via a light-addressable potentiometric sensor. The rate of acidification was then calculated from
the slope of a line fitted to the sensor data. The saline used was a
low-buffering capacity medium consisting of 138 mM NaCl, 5 mM
KCl, 0.81 mM K2HPO4, 0.11 mM KH2PO4, 0.5 mM MgCl2, 1.3 mM
CaCl2, and 5 mM glucose. The pH of all solutions was adjusted to a
value of pH 7.6 just before use in the cytosensor microphysiometer to
avoid possible modifications in acid secretion of the cells caused by
changes in extracellular pH. Anisotonic conditions were created by
variation of the NaCl concentration in the standard medium. The
experimental protocol was as follows. First, the cells were left to
recover from embedding for at least 1 h. A baseline value of acid
secretion was then determined, followed by a switch to either identical
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CYTOSKELETON AND VOLUME REGULATION IN HEPATOCYTES
presence of 7.2 ␮M of the calcium ionophore ionomycin, were determined to calibrate the measurements.
Statistics. Data are presented as means ⫾ SE of the number (n) of
independent preparations or individual cells. In the latter case, at least
three cell cultures obtained from three different fish were used.
Statistical differences were evaluated by applying analysis of variance
(ANOVA) followed by the appropriate post tests. A value of P ⬍ 0.05
was considered significant.
RESULTS
Fig. 1. Confocal fluorescence images of cytoskeletal
elements of trout hepatocytes. Top: 3-dimensional
reconstruction of the F-actin cytoskeleton, obtained
by merging 1-␮m optical slices, of control hepatocytes (A) and of cells incubated with 2 ␮M cytochalasin B (CB) for 30 min (B). Middle: microtubular
meshwork of a control hepatocyte (C) and a cell
pretreated with 100 ␮M colchicine for 30 min (D).
Images were obtained from an optical slice (1 ␮m
thick) through the equatorial plane of the cells. Bottom: optical slice through a cell costained for visualization of F-actin (E) and microtubules (F), and an
overlay of both images (G).
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Effect of cytoskeleton disrupting agents on F-actin and
tubulin. Staining of the actin network in control hepatocytes
revealed a mainly cortical localization of F-actin and a relatively even, low level of stained actin in the cytoplasm.
Three-dimensional images reconstructed from 1-␮m optical
slices through the cells thus yielded a rather smooth, spherical
appearance of the hepatocytes (Fig. 1A). Microtubular staining
of control cells yielded a fine meshwork of tubules across the
cytoplasm and clearly excluded the nuclear area (Fig. 1C).
Images obtained by dual labeling of actin and tubulin showed
a clear separation of both elements but indicated that at the cell
periphery, microtubules may be in contact with cortical actin
(Fig. 1, E–G). Incubation of trout hepatocytes with CB resulted
in a disruption of actin filaments that was often visible as a
disruption of the cortical actin ring and generally resulted in a
punctuate staining pattern of F-actin in three-dimensional projections of the cells (Fig. 1B). Qualitatively similar images
were obtained from cells incubated with 2 ␮M CD, the CB
analog, or with 2 ␮M latrunculin B (not shown). After exposure to colchicine for 30 min, microtubules still remained
visible, but the associated fluorescence was diminished to a
large extent (Fig. 1D).
Quantitative changes of F-actin distribution caused by CB
and during anisotonic exposure. The distribution of F-actin
was assessed by calculating the ratio between actin-fluores-
CYTOSKELETON AND VOLUME REGULATION IN HEPATOCYTES
cence from the cortical and the cytoplasmic region of the cells.
An example depicting a fluorescence profile obtained from an
individual hepatocyte is shown in Fig. 2A. After exposure to 2
␮M CB for 30 min, this method indicated a reduction of the
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cortical/cytoplasmic actin ratio to 78 ⫾ 3% of that determined
in control hepatocytes (Fig. 2B). During incubation with hypotonic medium, the cortical actin ring was reduced, both in
thickness and in its fluorescence intensity (Fig. 2, E and G).
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Fig. 2. Qualitative and quantitative assessment of
cellular F-actin distribution in trout hepatocytes
under control conditions, after treatment with CB,
and after exposure to hypo- or hypertonic saline. A:
image showing an optical section through the equatorial plane of a control cell (top) and the respective
fluorescence profile used to quantify the distribution of cortical actin relative to central actin (bottom). B: normalized ratio of cortical actin fluorescence to that of central actin for hepatocytes under
control conditions (isotonic saline, 284 mosM),
after 30 min of exposure to CB, after 5 and 30 min
of incubation in hypotonic saline (HYPO 5 and
HYPO 30, 166 mosM), and after 5 and 30 min of
incubation in hypertonic saline (HYPER 5 and
HYPER 30, 465 mosM). Data are expressed as
percentages of the mean of the control and are
presented as means ⫹ SE of 30 cells for each
condition. *P ⬍ 0.05 compared with controls.
C–H: representative images of cells subjected to
the indicated treatments and subsequently stained
for F-actin. Cont, control.
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CYTOSKELETON AND VOLUME REGULATION IN HEPATOCYTES
Quantitatively, this corresponded to a significant reduction of
the cortical/cytoplasmic actin ratio to 80 ⫾ 4 and 84 ⫾ 3% of
isotonic controls after 5 and 30 min of anisotonic exposure,
respectively (Fig. 2B). In contrast, no visible or quantitative
Table 1. Effect of CB (cell cultures), CD (cell suspensions), and cariporide on regulatory volume
changes of hepatocytes exposed to hypo- or hypertonic saline
Hypotonic Saline
Controls
CB/CD
Hypertonic Saline
Cariporide
Controls
CB/CD
2.36⫾0.26
38.6⫾6.3
73.5⫾5.8
0.41⫾0.18*
18.4⫾5.3*
43.5⫾6.0*
5.91⫾1.33
37.7⫾3.2
42.6⫾3.0
3.20⫾1.15
19.7⫾3.7*
24.2⫾4.6*
Cariporide
Cell cultures
VRVC
RVC20
RVC40
3.70⫾0.31
53.2⫾3.6
62.8⫾3.9
2.90⫾0.22
40.8⫾4.1
42.5⫾4.0*
Cell suspensions
VRVC
RVC20
RVC40
11.66⫾4.12
74.9⫾4.8
85.2⫾4.1
6.21⫾3.54
49.4⫾4.8*
61.9⫾4.8*
13.68⫾7.14
75.5⫾3.8
81.4⫾5.2
4.79⫾1.19
15.6⫾1.1*
15.8⫾3.7*
Cells were exposed to conditions as described in the legends to Figs. 3 and 4. VRVC describes the initial rate of volume recovery in % per min, calculated by fitting
data of regulatory volume change (RVC, %) vs. time (min) to the second-order polynomial A0 ⫹ vRVC t ⫹ Ct2. RVC20 and RVC40 denote the extent of regulatory
volume changes in %observed 20 min and 40 min after the maximum volume change, respectively. Data are means ⫾ SE of the number of cells and independent
experiments given in the legends to Figs. 3 and 4. *P ⬍ 0.05, significant differences compared with control cells. CB, cytochalasin B; CD, cytochalasin D.
AJP-Regul Integr Comp Physiol • VOL
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Fig. 3. Changes of relative cell water volume (Vr) of hepatocytes preincubated
with standard saline (284 mosM) in the absence or presence of 2 ␮M CB and
subsequently exposed to hypo- (166 mosM; A) or hypertonic conditions (465
mosM; B), as indicated. Cell water volume changes were measured using the
fluorescent dye calcein, applying an inverse epifluorescence microscope. At
the beginning of the experiments, a 3-point calibration was performed using
calibration media of 264 mosM (medium A), 308 mosM (medium B), and the
standard 284 mosM saline (medium C). The periods during which the respective media and cytochalasin were present is indicated in horizontal bars at
bottom. Data are means ⫾ SE of 60 cells from 4 independent experiments.
changes were detected in hypertonic saline (Fig. 2, F and H),
where the cortical/cytoplasmic actin ratio of hepatocytes remained constant at ⬃97% of the control value.
Changes of relative cell water volume. Changes of Vr in
response to hypo- and hypertonic exposure of hepatocytes are
depicted in Fig. 3; a summary of the parameters used to
characterize these alterations is given in Table 1. In control
hepatocytes, hypotonic exposure caused an increase of Vr to a
maximum of 1.67 ⫾ 0.02 after ⬃3 min (time 73 min in Fig.
3A), followed by a regulatory volume decrease (RVD) response of 53 ⫾ 3 and 63 ⫾ 4% after 20 and 40 min,
respectively. Addition of CB to the cells did not affect Vr under
isotonic conditions or Vrmax attained in hypotonic saline,
which amounted to 1.66 ⫾ 0.03 in these cells. However, CB
exposure affected subsequent cell volume recovery of the
hepatocytes, which showed a tendency to be reduced after 20
min and was diminished by 33% (P ⬍ 0.05) after 40 min. In
hypertonic saline, controls shrunk to a Vrmin of 0.62 ⫾ 0.02
after 2 min (time 72 min in Fig. 3B) and showed a regulatory
volume increase (RVI) of 39 ⫾ 6 and 73 ⫾ 6% after 20 and 40
min, respectively. Under these conditions, CB affected both
Vrmin, which amounted to 0.71 ⫾ 0.02 (P ⬍ 0.05), as well as
subsequent RVI, with all parameters determined being significantly reduced compared with untreated controls (Table 1).
Another series of experiments examined the impact of Factin disruption on volume regulation in suspended hepatocytes to clarify whether cell attachment would be a prerequisite
for the observed effects to occur. As shown in Fig. 4 and Table
1, the impact of CD on the regulatory volume changes of cells
in suspension was qualitatively very similar to the effect
exerted by CB on attached, cultured hepatocytes. Although in
hypotonic saline, maximal cell swelling (Vr ⫽ 1.29 ⫾ 0.03 and
1.30 ⫾ 0.16 in controls and CD-treated cells, respectively) was
reduced in suspended hepatocytes compared with attached
cells, CD nevertheless markedly inhibited RVD (Fig. 4A).
Similarly, CD significantly reduced hypertonic RVI of the
suspended hepatocytes (Fig. 4B). Interestingly, maximal cell
shrinkage was identical in controls (0.80 ⫾ 0.04, n ⫽ 10) and
CD-exposed cells (0.80 ⫾ 0.04, n ⫽ 7), ruling out the possibility that the slower rate of RVI of the latter was due to a
smaller stimulus for volume recovery.
CYTOSKELETON AND VOLUME REGULATION IN HEPATOCYTES
We also addressed the role of Na⫹/H⫹ exchange in the
volume regulatory response of the cells by following volume
recovery in the presence of the specific Na⫹/H⫹ exchanger
(NHE-1) inhibitor cariporide. This inhibitor did not significantly affect maximum cell swelling (1.37 ⫾ 0.16, n ⫽ 4) or
maximum cell shrinkage (0.78 ⫾ 0.03, n ⫽ 3). It also did not
affect RVD of the hepatocytes, but it dramatically reduced
RVI, which was virtually abolished after a slight initial volume
recovery during the first 10 min of hypertonic exposure (Fig.
4B, Table 1).
Cellular proton secretion under anisotonic conditions. Cellular proton secretion can be used as a measure for metabolic
activity of the cell, but it also provides information about the
activity of various ion transporters involved in both acid-base
regulation and osmolyte transport induced by anisotonic conditions. In fact, in control cells, exposure to hypotonic saline
evoked a rapid decrease of acid release by ⬃25%, and this
diminished rate of acid release persisted during 30 min of
hypotonic incubation (Fig. 5A). Incubating hepatocytes with
CB under isotonic conditions transiently increased acid secretion to 137 ⫾ 4% of the basal rate, but it returned to 104 ⫾ 2%
AJP-Regul Integr Comp Physiol • VOL
after 30 min of incubation (Fig. 5B). A subsequent switch to
hypotonic medium decreased the proton release rate by 28%,
which was not significantly different from the decrease observed in controls. Exposure to hypertonic medium evoked an
increase to 237% of the basal rate in controls, followed by a
reduction to 159%, at which level it stabilized after ⬃15 min of
incubation (Fig. 5C). In CB-treated cells, which again displayed the transient increase of proton release noted above,
hypertonicity elevated acid secretion to 229% of the basal rate
(P ⬎ 0.05 compared with control cells). However, in contrast
to untreated hepatocytes, the acidification rate did not stabilize
at an elevated level but returned to the basal level observed
before the hypertonic challenge after 18 min (Fig. 5D). In cells
incubated with CD, a nearly identical response was observed,
with an immediate acid secretion maximum of 271% following
a switch to hypertonic medium and a subsequent rapid recovery to the basal level within 18 min.
On the basis of our previous observation that Na⫹/H⫹
exchange is strongly activated in trout hepatocytes during
hypertonic exposure (37), we investigated how inhibition of
this transporter would affect the acid release pattern observed
in these conditions. As shown in Fig. 6, addition of the
Na⫹/H⫹ exchange inhibitor amiloride reduced acid secretion
to 76 ⫾ 4% of the initial value. More importantly, we observed
that this treatment completely abolished the increase of proton
release induced by hypertonic saline seen in the absence of the
inhibitor.
In a separate series of experiments, we investigated the
effect of the microtubule-disrupting agent colchicine. We observed that neither isotonic nor anisotonic rates of proton
release were to any appreciable extent affected by this agent
(Fig. 7).
Intracellular pH. As shown in Fig. 8A, in untreated hepatocytes, hypotonicity induced a decrease of pHi by 0.15 units
from a value of 7.36 ⫾ 0.03 at time 0 to 7.21 ⫾ 0.02 30 min
later. In principle, CB-treated cells followed a similar pattern
of hypotonic acidification, but in this case the initial and final
values of pHi were 7.18 ⫾ 0.02 and 7.09 ⫾ 0.02, respectively,
and the change of pHi (⌬pHi) amounted to 0.09 units. Because
hepatocytes had different starting pHi values under both treatments, we converted ⌬pHi to net H⫹ accumulated, neglecting
intracellular buffering capacity. This calculation yielded 12 ⫾
2 and 14 ⫾ 2 nmol of H⫹ for controls and CB-treated cells,
respectively (P ⬎ 0.05). As evaluated separately, the lower
initial pHi was due to CB, which caused a slowly progressing
intracellular acidification in isotonic saline (Fig. 8A, inset).
In hypertonic medium, pHi of control cells increased from a
starting value of 7.39 ⫾ 0.02 to a peak value of 7.76 ⫾ 0.03
after 13 min, with ⌬pHi amounting to 0.37 units (Fig. 8B). The
alkalinization started immediately after the hypertonic shock
and slightly declined again after the maximum. In contrast, in
CB-treated cells (pHi at time 0 ⫽ 7.24 ⫾ 0.03), pHi showed a
slight initial decrease after the switch to hypertonic medium
and subsequently displayed a biphasic response, with a more
rapid increase in the first 10 min and a slower alkaline drift
during the next 20 min (pHi at 30 min ⫽ 7.42 ⫾ 0.04). The
⌬pHi calculated for the entire period amounted to 0.17 units.
After preincubation with latrunculin, cells had a starting pHi of
7.32 ⫾ 0.02 and reached a maximum pHi of 7.54 ⫾ 0.03 after
⬃10 min, corresponding to a ⌬pHi of 0.22 units. Converting
⌬pHi values to the net amount of H⫹ extruded, we obtained
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Fig. 4. Changes of Vr of hepatocytes maintained in suspension preincubated
with standard saline (284 mosM) in the absence (control) or presence of 2 ␮M
cytochalasin D (CD) or after addition of the Na⫹/H⫹ exchanger (NHE-1)
inhibitor cariporide and subsequently exposed to hypo- (166 mosM; A) or
hypertonic conditions (465 mosM; B). Volume changes were determined by
electronic cell sizing on a Coulter Counter Multisizer II. Data are means ⫾ SE
of 4 –5 independent experiments in hypotonic saline and of 10 (control), 7
(CD), and 3 (cariporide) independent experiments in hypertonic conditions.
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CYTOSKELETON AND VOLUME REGULATION IN HEPATOCYTES
24 ⫾ 2, 20 ⫾ 4, and 19 ⫾ 2 nmol H⫹ in controls, CD-treated
cells, and latrunculin-treated cells, respectively (P ⬎ 0.05).
Prompted by our findings on proton secretion rates, we
suspected that these different pHi response patterns could have
been caused by an effect of cytoskeletal disruption on the
Na⫹/H⫹ exchanger. To investigate this possibility, we examined the pHi recovery in controls and CB-treated hepatocytes
after the induction of an acid load, a condition known to
strongly activate this ion transporter in trout hepatocytes (23,
60, 63). Intracellular acidification was achieved using the NH⫹
4
prepulse technique (8, 47) with concurrent addition of the
inhibitor of Na⫹-dependent Cl⫺/HCO⫺
3 exchange, SITS, to
prevent compensatory activation of this transporter should
Fig. 6. Proton release of trout hepatocytes before and after block of the
Na⫹/H⫹ exchanger with 1 mM amiloride and during subsequent exposure to
hypertonic conditions. Data are means ⫾ SE of 12 independent experiments.
AJP-Regul Integr Comp Physiol • VOL
Na⫹/H⫹ exchange indeed have been diminished by the CB
treatment. As depicted in Fig. 9, addition of NH⫹
4 resulted in a
pronounced alkalinization and, after the washout of extracellular NH⫹
4 , a rapid acidification of pHi (⌬pHi ⫽ 0.25 compared
with the resting value). In controls, this was followed by
gradual recovery of pHi toward the resting level. In comparison, in CB-treated hepatocytes, a slightly but not significantly
larger increase of pHi was induced by addition of NH⫹
4.
However, a much less pronounced acidification was noted
upon removal of extracellular NH⫹
4 (⌬pHi ⫽ 0.09), and there
was no pHi recovery to be noted subsequently.
Similar to our findings on acid secretion rates, preincubation
of hepatocytes with colchicine had no significant effects on pHi
under isotonic conditions and also did not alter pHi changes
evoked by anisotonic conditions (data not shown).
Cytosolic free Na⫹ concentration. To validate our method
employed for the determination of [Na⫹]i, we initially exposed
hepatocytes to the Na⫹-K⫹-ATPase inhibitor ouabain. As to be
expected, this caused Na⫹ to slowly accumulate in the cells,
starting from a basal level for [Na]i of 19 mM to a value of 44
mM after 30 min of incubation with ouabain (Fig. 10, inset). In
isotonic saline, we noted a slight increase of [Na⫹]i in controls
from 16 ⫾ 1 mM at time 0 to 19 ⫾ 1 mM after 30 min. During
incubation with CB, this increase was slightly but significantly
more pronounced, elevating [Na⫹]i from 17 ⫾ 1 mM to 22 ⫾
1 mM within this period. The impact of hypertonic conditions
on [Na⫹]i is depicted in Fig. 10. In control cells, hypertonicity
caused an immediate increase of [Na⫹]i to 24 ⫾ 1 mM within
the first minute of exposure to a peak at 32 ⫾ 1 mM after ⬃8
min and a slight subsequent decrease to 30 ⫾ 1 mM after 30
min. In hepatocytes pretreated with CB, the initial increase of
[Na⫹]i was significantly larger and reached 29 ⫾ 1 mM after
1 min, followed by a further gradual increase to a final value of
47 ⫾ 1 mM at the end of the 30-min period of investigation.
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Fig. 5. Proton release of agarose-embedded
hepatocytes determined with a cytosensor
microphysiometer, with a light-addressable
potentiometric sensor that continuously
measured the rate at which the cells acidify
their environment. Cells were exposed to
hypo- (A and B) or hypertonic conditions (C
and D) after 30 min preexposure to either
standard saline (A and C) or to 2 ␮M CB (B
and D). Open circles in D depict cells before
and after 15 min of incubation with CD.
Data are means ⫾ SE of ⱖ6 independent
experiments.
CYTOSKELETON AND VOLUME REGULATION IN HEPATOCYTES
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Fig. 8. Changes of intracellular pH (pHi) of hepatocytes exposed to hypo- (A)
and hypertonic saline (B) and the effect of preincubation with CB or latrunculin B (Lat; hypertonic saline only). Anisotonic conditions were established
at time 0, as indicated by arrows. The lower initial pHi in CB-pretreated cells
was a consequence of the preincubation with the drug (A, inset; arrow indicates
addition of CB). pHi was determined by fluorescence microscopy using the
fluorescent dye 2⬘,7⬘-bis-(2-carboxypropyl)-5(6)-carboxyflourescein (BCPCF)
AM. Data are means ⫾ SE of ⱖ30 cells from ⱖ3 independent preparations
exposed to each condition.
Fig. 7. Proton release of trout hepatocytes exposed to hypo- (A) or hypertonic
conditions (B) after 30-min preexposure to either standard saline (control) or to
100 ␮M colchicine (COL). Data are means ⫾ SE of ⱖ4 independent experiments.
AJP-Regul Integr Comp Physiol • VOL
respectively, and the difference, i.e., Rb⫹ uptake mediated by
Na⫹-K⫹-ATPase, amounted to 0.48 ⫾ 0.03 nmol䡠106
cells⫺1 䡠min⫺1. In comparison, after 30 min of incubation with
CB, total Rb⫹ uptake was significantly reduced to 0.25 ⫾ 0.08
nmol䡠106 cells⫺1 䡠min⫺1 (n ⫽ 5), whereas ouabain-resistant
uptake was slightly increased to 0.11 ⫾ 0.05 nmol䡠106
cells⫺1 䡠min⫺1 (P ⬎ 0.05). The active rate of Rb⫹ uptake
therefore amounted to 0.14 ⫾ 0.13 nmol䡠106 cells⫺1 䡠min⫺1,
which corresponds to a 71% reduction compared with the
controls.
Cytosolic free Ca2⫹ concentration. Finally, we investigated
the effect of CB on changes of [Ca2⫹]i elicited by anisotonic
challenges, an important element in the volume regulatory
response of various cells. In line with previous observations
(37), we found that hypotonic exposure evoked only a slight,
gradually developing elevation of [Ca2⫹]i in trout hepatocytes,
and neither the pattern nor the extent of this increase was
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As shown below, CB significantly diminished Na⫹-K⫹ATPase activity. Therefore, we tested whether the elevated
increase of [Na⫹]i in CB-treated cells could have been a result
of this inhibition. As shown in Fig. 10, [Na⫹]i of ouabaintreated cells increased to 20 ⫾ 2 mM and further to 27 ⫾ 2 mM
after 1 and 2 min of hypertonic exposure, respectively. Thereafter, [Na⫹]i indeed showed an increase similar to that noted in
CB-treated cells, with [Na⫹]i reaching a final value of 52 ⫾ 3
mM at 30 min. We also examined the possibility that reduced
Na⫹/H⫹ exchanger activity could underlie the elevated [Na⫹]i.
However, the presence of cariporide did not alter the increase
of Na⫹, with [Na⫹]i reaching a value of 23 ⫾ 1 after 1 min, a
first plateau at 31 ⫾ 1 mM after 5 min, and a final value of
35 ⫾ 2 mM after 30 min (n ⫽ 51 cells from 3 independent
preparations).
Na⫹-K⫹-ATPase activity. Another series of experiments
addressed the impact of cytoskeletal disruption on both the
primary active uptake of K⫹ via Na⫹-K⫹-ATPase activity as
well as on ouabain-resistant uptake of K⫹. In control cells, the
rates of total and ouabain-resistant Rb⫹ uptake amounted to
0.53 ⫾ 0.05 and 0.04 ⫾ 0.03 nmol䡠106 cells⫺1 䡠min⫺1 (n ⫽ 6),
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CYTOSKELETON AND VOLUME REGULATION IN HEPATOCYTES
Fig. 9. Changes of pHi of trout hepatocytes, after 30 min of
incubation in the absence (control) or presence of 2 ␮M CB,
induced by the addition and removal of 30 mM NH4Cl, and
subsequent pHi recovery. The period of NH4Cl exposure is
indicated by the horizontal bar. Both the NH4Cl medium and
the washing saline contained the Na⫹-dependent Cl⫺/HCO⫺
3
exchange inhibitor 4-acetamido-4⬘-isothiocyanostilbene-2,2⬘disulfonic acid (SITS) at a concentration of 0.5 mM. Data are
means ⫾ SE of ⱖ30 cells from 3 independent preparations.
Fig. 10. Alterations of cytosolic free Na⫹ concentration ([Na⫹]i) induced by
hypertonic conditions after 30 min of preincubation in control saline or with
CB and in cells treated with ouabain (Ob) for 5 min. Cells were exposed to
anisotonic conditions at time 0, as indicated by the arrow. [Na⫹]i was measured
with the Na⫹-sensitive dye SBFI AM using a fluorescence microscope. Data
are means ⫾ SE of 75– 80 cells from 3– 4 independent preparations. Inset:
effects of addition of Ob on [Na⫹]i in hepatocytes during 30 min of incubation
with the drug. Data are means ⫾ SE of 13 cells from 2 independent
preparations.
AJP-Regul Integr Comp Physiol • VOL
DISCUSSION
The present study provides various lines of evidence that the
actin-based cytoskeleton plays an important, multifaceted role
for cell volume regulation of trout hepatocytes. We found that
the distribution of actin filaments remained unaffected under
hypertonic conditions but was significantly altered during hypotonic exposure of the cells. Within 5 min of hypotonic
incubation, cortical actin was significantly reduced and showed
only a slight tendency for recovery during 30 min. Quantitatively, these changes corresponded to a 15–20% reduction in
the ratio of cortical to central F-actin fluorescence (P ⬍ 0.05)
during this period. This relative decrease of cortical F-actin
agrees with similar observations on various other cells (26, 54),
including chondrocytes, where the same method was used (17).
Fig. 11. Changes of intracellular free Ca2⫹ concentration ([Ca2⫹]i) induced by
hypertonic conditions after 30 min of preincubation in control saline or with
CB. Anisotonic conditions were created at time 0, as indicated by the arrow.
[Ca2⫹]i was measured in fura-2-loaded cells using fluorescence microscopy.
Data are means ⫾ SE of ⱖ40 cells from 4 independent preparations.
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significantly affected by CB (data not shown). In contrast, in
hypertonic saline, control hepatocytes showed a rapid increase
of [Ca2⫹]i from an initial level of 92 ⫾ 11 nM to a peak of
160 ⫾ 20 nM after 6 min, where [Ca2⫹]i was maintained with
some fluctuations during the rest of the experimental period
(Fig. 11). In CB-treated cells, baseline [Ca2⫹]i was 57 ⫾ 6 nM
at time 0 and increased to 89 ⫾ 9 nM upon hypertonic
exposure, with this elevation being significantly lower than in
controls. Thereafter, this elevated value remained relatively
constant.
CYTOSKELETON AND VOLUME REGULATION IN HEPATOCYTES
AJP-Regul Integr Comp Physiol • VOL
this inhibition impaired the RVD response. Accordingly, the
intracellular acidification of CB-treated hepatocytes following
hypotonic exposure was not altered compared with control
cells, yielding similar amounts of H⫹ accumulated over 30
min. The relatively minor role of acid-transporting pathways in
RVD is also underscored by the lack of any effect of Na⫹/H⫹
exchange inhibition on volume recovery. Although this may be
typical for many cells, activation of Na⫹/H⫹ exchange upon
swelling has in fact been observed in certain cell types (46) and
under certain conditions (51).
In contrast to hypotonic conditions, during hypertonic exposure, CB treatment reduced cell shrinkage in attached cells
compared with controls but did not alter that of cells in
suspension. The reasons underlying this discrepancy remain
unknown at present. However, our data show that in both cell
cultures and suspended hepatocytes, a significantly diminished
extent of volume recovery after 20 and 40 min can be observed. This finding suggests that in neither hypotonic nor
hypertonic conditions is cell attachment prerequisite for an
effect of F-actin disruption on volume regulatory processes. In
the latter case, our study provides direct evidence that this is
due to an interaction of the actin-based cytoskeleton and the
activity of ion transporters. As an outstanding candidate, we
identified the Na⫹/H⫹ exchanger, because its inhibition by
cariporide reduced RVI by ⬃60%. In control cells, hypertonic
saline evoked a pronounced increase of proton secretion, the
time course and pattern of which largely paralleled the changes
in cell volume. In addition, these changes were accompanied
by a substantial alkalinization, which, as previously shown (20,
37), is entirely due to enhanced activity of the Na⫹/H⫹ exchanger. This was further corroborated by showing that in the
presence of amiloride, hypertonic exposure did not result in an
increase of proton release. Recent experiments in which the
rather specific NHE-1 inhibitor cariporide was used confirmed
these findings. Cariporide completely inhibited the hypertonic
alkalinization, and it also blocked most, but not all, of the
hypertonicity-induced increase of proton release. This finding
suggests that other transporters mediating net acid secretion
were concurrently activated in hypertonic saline and that the
complete inhibition seen with amiloride was in part due to
nonspecific effects of the drug on these transporters (Ahmed K,
Pelster B, and Krumschnabel G, unpublished observation).
In CB-treated cells, the hypertonically induced increase of
proton release was only short-lived and pHi was transiently
even decreased, followed by a significantly delayed elevation
of pHi compared with the controls. In comparison, latrunculin
did not acidify hypertonically exposed cells, and it reduced the
subsequent increase of pHi. Expressed as H⫹ extruded, the
reduction in the intracellular alkalinization corresponded to a
decrease in proton secretion by ⬃20%. Thus we conclude that
the activity of the Na⫹/H⫹ exchanger is, at least in part, under
the control of the actin-based cytoskeleton, with an intact
cytoskeleton required for full activation of this transporter
under hypertonic conditions. In principle, this finding is in line
with other reports, although the best documented example in
this regard is the NHE-3 isoform of the Na⫹/H⫹ exchanger
family (40, 58), which is not involved in the RVI response,
because it is inhibited, rather than activated, by osmotic shrinkage (34). However, a close link between NHE-1, the housekeeping isoform related to regulation of cell volume and pHi in
most cells (62), and the cytoskeleton has recently been firmly
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The lack of an effect of hypertonicity on F-actin distribution
also has been reported for some other cells (17) but is at
variance with some studies in which an increase of F-actin has
been observed (25, 54, 57). These results clearly demonstrate
that the actin-based cytoskeleton is affected by cell volume
changes in many cells but that there may be cell type-specific
differences with respect to the exact mechanism of this modification (53).
In addition to the hypotonic effect on the actin-based cytoskeleton, we observed that disruption of the F-actin by use of
cytochalasins affected both cell volume changes elicited by
anisotonic challenges and ionic fluxes associated with volume
regulation in trout hepatocytes. Several previous studies failed
to document such effects, but at least in some cases this
appears to be due to technical difficulties. For example, the
concentration of CB or one of its analogs adequate to disrupt
F-actin in one cell type may be inadequate to disrupt the
F-actin in another cell line (15). Furthermore, cytochalasins
may even increase, instead of decrease, F-actin when used at
high concentrations (21). To exclude this possibility, we both
qualitatively and quantitatively assessed the impact of CB on
F-actin of trout hepatocytes. As shown in Figs. 1 and 2, the
mainly cortical localization of F-actin in control hepatocytes, a
feature typical for many cells, was clearly disrupted by CB at
the concentration and the preincubation period applied. As a
result of this, the cortical/cytoplasmic actin ratio was diminished by ⬃20% compared with the control value.
Although quantitatively this decrease corresponded to that
observed in hypotonically exposed cells, its physiological effect was quite dissimilar, leading to an inhibition of RVD. The
actin rearrangement induced by cell swelling therefore seems
to be a more subtle and controlled process, which may either
not affect or possibly even enhance volume regulatory responses. In contrast, the rather nonspecific disruption of F-actin
by CB appears to destroy structures required for normal cell
volume regulation and therefore does not mimic the hypotonic
alterations. In line with this finding, F-actin disruption significantly reduced RVI of hepatocytes, although we could not
detect a change of F-actin distribution under hypertonic conditions. Thus the integrity of F-actin may not directly control,
but could act as a permissive factor for, the function of
transporters involved in volume regulatory processes.
In agreement with this notion, neither maximum swelling
nor the initial rate of volume recovery was affected by CB or
CD, which is consistent with previous reports (15, 19, 54).
Subsequently, however, CB and CD delayed volume recovery,
suggesting a possible link between actin filaments and volume
regulatory transporters, e.g., K⫹ and Cl⫺ exit pathways, which
become activated upon swelling in many cells (30, 41), including trout hepatocytes (6, 52). An interaction between F-actin
and these transporters has been previously documented for
mammalian cells (9, 33, 50). In the present study, we concentrated on proton-linked ion transport pathways, and therefore
we can only speculate on the possible connection between the
actin-based cytoskeleton and K⫹ and Cl⫺ exit pathways that do
not involve the net transport of acidic equivalents. Although
disruption of F-actin significantly attenuated the RVD response
of hepatocytes compared with control cells, the decrease in the
rate of acid secretion during hypotonic exposure was not
affected by CB. This finding clearly indicated that CB inhibited
ion release of hepatocytes independent of proton release, and
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osmotically relevant ion transport mechanisms that do not
transport acid or base equivalents in parallel.
Besides these direct effects of Na⫹/H⫹ exchange on osmotic
and cell volume regulation, it should be pointed out that an
inhibition of this transporter may elicit indirect effects. Efflux
pathways for K⫹ and Cl⫺ involved in RVD have been found to
be inhibited at low pH (31), which could in part account for the
slowed RVD of cytochalasin-treated cells. On the other hand,
Na⫹/H⫹ exchange activity is stimulated at low pH, and thus
the decreased pHi should accelerate RVI and not inhibit it. To
clarify this matter, investigation of the pH dependence of
volume recovery in trout hepatocytes is currently under way.
A final aspect we addressed in the present study was the
potential impact of cytoskeletal disruption on [Ca2⫹]i changes
evoked by anisotonic exposure. Although the importance of
Ca2⫹ in volume regulation is not fully understood, several
studies indicate an important role, and more importantly, there
is evidence for functional interactions between intracellular
Ca2⫹ and the cytoskeleton (7, 32). With regard to the latter,
Erickson et al. (17) recently showed that, in chondrocytes,
F-actin redistribution evoked by hypotonic exposure was
strictly dependent on [Ca2⫹]i oscillations induced in the presence of extracellular Ca2⫹. Although this was not tested in the
present study, we saw that hypotonicity induced an increase of
[Ca2⫹]i and a redistribution of F-actin but that disruption of the
cytoskeleton had no effect on [Ca2⫹]i changes. At the same
time, however, the CB treatment significantly diminished the
[Ca2⫹]i increase elicited in hypertonic saline. The importance
of intracellular Ca2⫹ in the RVI response of trout hepatocytes
is not known, but at least the NHE-1 isoform of Na⫹/H⫹
exchangers is known to be stimulated by an increase of [Ca2⫹]i
via a cytoplasmic calmodulin binding site, the deletion of
which drastically inhibits its hypertonicity-induced activation
(4, 61).
Of further note is that none of the parameters investigated
appeared to be in any way significantly affected by colchicine
incubation, despite the fact that our visual inspection indicated
that the microtubular meshwork was indeed disrupted by this
treatment. Therefore, in agreement with other studies (11, 12),
we conclude that intact microtubules are not a prerequisite for
normal cell volume regulation, at least not during short-term
anisotonic exposure.
GRANTS
This work was supported by FWF Austria (P16154-B06) and F. Antorchas,
University of Buenos Aires, CONICET, ANPCYT (01-11017).
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