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Biophys J, April 2001, p. 1783-1790, Vol. 80, No. 4
and
*Forschungsinstitut für Molekulare Pharmakologie, D-10315
Berlin, Carl Zeiss, Mikroskopie, Vertrieb Berlin, D-10787
Berlin, and Freie Universität Berlin, Institut
für Pharmakologie, D-14195 Berlin, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The water channel aquaporin-2 (AQP2), a key component of the antidiuretic machinery in the kidney, is rapidly regulated by the antidiuretic hormone vasopressin. The hormone exerts its action by inducing a translocation of AQP2 from intracellular vesicles to the cell membrane. This step requires the elevation of intracellular cyclic AMP. We describe here a new method, laser scanning reflection microscopy (LSRM), suitable for determining cellular osmotic water permeability coefficient changes in primary cultured inner medullary collecting duct (IMCD) cells. The recording of vertical-reflection-mode x-z-scan section areas of unstained, living IMCD cells proved useful and valid for the investigation of osmotic water permeability changes. The time-dependent increases of reflection-mode x-z-scan section areas of swelling cells were fitted to a single-exponential equation. The analysis of the time constants of these processes indicates a twofold increase in osmotic water permeability of IMCD cells after treatment of the cells both with forskolin, a cyclic AMP-elevating agent, and with Clostridium difficile toxin B, an inhibitor of Rho proteins that leads to depolymerization of F-actin-containing stress fibers. This indicates that both agents lead to the functional insertion of AQP2 into the cell membrane. Thus, we have established a new functional assay for the study of the regulation of the water permeability at the cellular level.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Water permeability across biological membranes
has been extensively investigated. Because pure lipid bilayer membranes
are weakly permeable to water, pore-like structures facilitating the water transport across bilayered biological membranes were postulated first in the 1950s (Koeford-Johnson and Ussing, 1953
). The first proof
for a protein acting as a membrane spanning water-selective pore was
provided by Preston et al. (1992), using Xenopus oocytes microinjected with mRNA encoding aquaporin-1, originally named CHIP-28.
In contrast to electrophysiological measurements of ion currents, which
allow high time and signal resolution at the level of a single cell and
even a single channel, the techniques available for the measurement of
water transport across cell membranes are restricted due to the
uncharged nature of water molecules. For example, the determination of
water flow across artificial membranes or intact epithelia such as
freshly isolated single nephron fragments in microperfusion experiments
(Burg et al., 1966) had to be performed using tritiated water. At the
single-cell level, the measurement of water permeabilites was performed
by monitoring an indirect parameter, the cell volume, determined after
three-dimensional reconstruction of the cell shape (e. g. Fisher et
al., 1981). Although this method afforded accurate cell volume
determination it was relatively slow. To improve the time resolution,
approaches to determine water transport across cell membranes of
adherent single cells using two types of markers were introduced. In
the first approach, fluorescent microbeads were fixed with gelatin on
the culture dishes before seeding of epithelial cells and again applied
before the experiment to the apical membranes of the cells grown in the
pretreated culture dishes. The change in the distance separating the
apically localized beads from those on the dishes served as a
one-dimensional measure of cell volume changes after an osmotic
challenge (Crowe and Wills, 1991). This technique was improved by van
Driessche et al. (1993) using an automatically focusing bead-tracking
device driven by a piezoelectric motor to move a microscope objective.
Second, to measure changes in cell volume, the fluorescent dye
calcein-AM was entrapped within the cells and analyzed by total
internal reflection microflourimetry. An increase in cell volume
results in a decreased fluorescence signal from the cytosol, whereas
increasing fluorescence intensity indicates cell shrinkage (Farinas et
al., 1995). Recently a new technique, scanning ion conductance
microscopy (Korchev et al., 2000), has been described, which allows
high time resolution (milliseconds) for measurement of cell height
changes but requires the addition of piezo stepper-driven equipment and
microelectrode amplifiers to the microscope. In this study, a
comparison of cell height measurement by scanning ion conductance
microscopy with the determination of cell volume changes by preloaded
fluorophores showed comparable results.
We present a novel optical method to continuously monitor cell volume
changes using a confocal laser scanning microscope. Instead of using
preloaded cellular indicators, we applied the so-called reflection mode
of confocal laser scanning microscopy to visualize vertical sections of
unstained, living cells. Because of the different optical densities of
the coverslip glass, the intracellular fluids and the bilayered cell
membrane, the laser beams are reflected at these optical borders and
return through the objective to the photomultiplier as in fluorescence
microscopy (see Fig. 1). Thus, the movement of the plasma membranes of
swelling or shrinking cells can be tracked directly by reflected laser beams. The parameter used to determine the kinetics of swelling or
shrinking cells by laser scanning reflection microscopy (LSRM) is a
two-dimensional optical vertical section through the cells. It is
noteworthy to mention that LSRM is different from the reflection interference contrast microscopy (e.g., Filler and Peuker, 2000), which
uses optical phenomena caused by oblique epi-illumination from a
monochromatic light source.
Primary cultured inner medullary collecting duct (IMCD) cells are a
cellular model consisting mainly of vasopressin-sensitive principal
cells responsible for water reabsorption within the collecting duct.
Cultured IMCD cells endogenously express the vasopressin-regulated
antidiuretic machinery, in particular the vasopressin V2 receptor and
the water channel aquaporin-2 (AQP2) (Maric et al., 1998). Incubation
of IMCD cells with cAMP-elevating agents (arginine-vasopressin or
forskolin) induces the translocation of AQP2 to the cell membrane,
similar to principal cells in vivo. In addition, incubation of the
cells with Clostridium difficile toxin B, which leads to
depolymerization of F-actin-containing stress fibers by inhibiting
members of Rho-family proteins (Hall, 1998; Lerm et al., 2000), has
been shown to induce hormone independently the translocation of AQP2 to
the cell membrane (Klussmann et al., 2001). The hormone-induced
trafficking of AQP2 was described as the shuttle hypothesis (Wade et
al., 1981).
We have applied LSRM to study the effects of forskolin and toxin B on the regulation of osmotic water permeability in IMCD cells. Osmotic water permeability changes could be induced by the cAMP-elevating agent forskolin and hormone independently by toxin B. The specificity of the effect of C. difficile toxin B on primary cultured IMCD cells was verified by application of the toxin on Madin-Darby canine kidney (MDCK) cells, which are void of the above mentioned proteins of the antidiuretic machinery.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell culture
IMCD cells were prepared and cultured as described (Maric et
al., 1998). Briefly, Wistar rats were killed by decapitation, and
kidney inner medullae (including papillae) were removed and cut into
small pieces. Tissue was digested in phosphate-buffered saline (PBS:
137 mM NaCl, 2.7 mM KCl, 1 mM
KH2PO4, 10 mM
Na2HPO4, pH 7.4) containing
0.2% hyaluronidase (Boehringer Mannheim, Mannheim, Germany) and 0.2%
collagenase type CLS-II (Biochrom, Berlin, Germany) at 37°C for 90 min. Thereafter cells were centrifuged (5 min; 300 × g), washed three times, and seeded at a density of
approximately 7 × 104
cells/cm2 on glass coverslips (30 mm diameter)
coated with type IV collagen (2 µg/cm2;
Becton-Dickinson, Heidelberg, Germany) in petri dishes. Because IMCD
cells are accustomed to high osmotic challenges within the kidney
medulla, Dulbecco's modified Eagle's medium (DMEM), adjusted to 600 mOsm/kg by addition of 100 mM NaCl and 100 mM urea, was used to
establish growth conditions with preferential selectivity for IMCD
principal and intercalated cells (Mooren and Kinne, 1994). Penicillin
(100 IU/ml), streptomycin (100 µg/ml), glutamine (2 mM), nonessential
amino acids (1%), fetal calf serum (10%), and dibutyryl cyclic AMP
(DBcAMP; 500 µM) were routinely added. The cell culture medium was
exchanged three times per week. Approximately 18 h before the
experiments, the medium was replaced by cell culture medium without
DBcAMP to render the cells readily stimulable by cAMP-elevating agents.
MDCK cells (strain II) were kept in DMEM (osmolality of 300 mOsm/kg) containing penicillin (100 IU/ml), streptomycin (100 µg/ml), and fetal calf serum (10%). MDCK cells were used from passage 48 to passage 52.
Fluorescence microscopy and visualization of F-actin
For fluorescence microscopy cells were grown on glass coverslips
(12 mm diameter) as described above. IMCD and MDCK cells were left
untreated or treated with C. difficile toxin B in different concentrations (4 µg/ml to 4 ng/ml) to estimate appropriate
concentrations of the toxin for both cell types (i.e., depolymerization
of stress fibers). For visualization of the F-actin-containing stress
fibers, both cell types were fixed and permeabilized as described
(Maric et al. 1998). They were incubated with phalloidin-TRITC (100 µg/ml) for 30 min and subsequently washed with PBS. Fluorescence
signals were analyzed by epifluorescence microscopy (Leica DMLB
microscope with cooled Sensicam 12 bit CCD camera, Bensheim, Germany).
Measurements of cell volume kinetics by LSRM x-z-scan time series
Bathing solutions
The above described cell culture media were used as experimental bathing solutions with two exceptions: 1) instead of the bicarbonate/CO2-based buffer system, HEPES-buffer (10 mM) served to maintain a constant pH of 7.4 under room air conditions, and 2) fetal calf serum was replaced by sorbitol to adjust the osmolality to 600 mOsm/kg for IMCD cells and to 300 mOsm/kg for MDCK cells. These normotonic (N) bathing solutions were diluted with distilled water to obtain hypotonic (H) solutions. For validation experiments on IMCD cells (see Results), solutions with osmolalities from 100 up to 900 mOsm/kg were used. The solutions with higher osmolalities (750 mOsm/kg) were obtained by further addition of NaCl and urea (50 mM each) and solutions with an osmolality of 900 mOsm/kg by addition of 100 mM of each of these compounds. For functional experiments determining Pf changes, only N and H solutions with osmolalities of 600 and 200 mOsm/kg, respectively, were used for IMCD cells, whereas MDCK cells were bathed in N and H solutions with osmolalities of 300 and 100 mOsm/kg, respectively. The osmotic challenge for both cell types was therefore considered to be identical (threefold dilution of the appropriate solution osmolalities).Preparation of cells for cell volume kinetics measurements
In primary cultured IMCD cells, the vasopressin-regulated water channel AQP2 is sorted mainly to the lateral (rather than the apical) membranes upon stimulation with forskolin or vasopressin (Maric et al., 1998; Klussmann et al., 1999, 2000). To ensure free access of bathing
solutions to the lateral membranes, IMCD cells were artificially made
noncoherent by trypsinization (37°C; 8 to 12 min with 0.05%
trypsin/0.02% EDTA; Biochrom). After being allowed to recover from the
trypsinization procedure for at least 2 h under growth conditions,
the IMCD cells were washed twice with bathing solution N (600 mOsm/kg).
The coverslip with the cell layer was carefully removed from the
culture dish and mounted for the entire experiment in a custom-made
cuvette fitted into the motor-driven x-y table of
the laser scanning microscope (LSM 410, Zeiss, Jena, Germany).
MDCK cells were cultured until they reached a subconfluent density,
washed twice with the appropriate bathing solution N (300 mOsm/kg), and
mounted as described for IMCD cells.
Cell swelling reaction and data acquisition
Appropriate cells, i.e., individualized cells that morphologically resembled principal cells and to whose lateral membranes solute had free access, were selected using the transmission mode of the LSM 410 (Fig. 1). Next, the optical setting of the LSM 410 was changed to the reflection mode using a long-pass emission filter of 515 nm, thus allowing the recording of the reflected laser beam with 543 nm wavelength (Fig. 1). In contrast, an appropriate setting for fluorescence microscopy would use a long-pass emission filter of 570 nm to allow for the recording of the light emitted by a fluorophore (e.g., TRITC;
= 582 nm) that is
excited by the laser with a wavelength of = 543 nm (Fig. 1).
Cells were scanned with a ×63 magnification water immersion objective,
numerical aperture 1.2. The initially applied bathing solution N (600 mOsm/kg for IMCD cells and 300 mOsm/kg for MDCK cells) was removed by suction using a peristaltic pump, leaving only a very thin film of
solution (~20 µm) above the cells. Cell swelling was then initiated by the application of bathing solution H (200 mOsm/kg for IMCD cells
and 100 mOsm/kg for MDCK cells). Customized macro-programming facilitated the recording of reflection-mode
x-z-scans with a frequency of 0.25 Hz;
x-z-scans were stored as time series. All recordings were started 40 s before the change of N to H bathing solution and continued for 200 s. The osmotic challenges were performed and monitored on the microscope at elevated room temperature (27°C). Thereafter, cells were allowed to recover from the osmotic challenge in bathing solution N. For this purpose they were placed inside the metal cuvette on a precision heating block (33°C;
Precitherm, Duesseldorf, Germany) under occlusion to prevent
evaporation and thus uncontrolled increase of the osmolality of the
bathing solution. During this period (up to 3 h 30 min), cells
were either left untreated or stimulated as indicated in the Results.
Recovery or incubation of the cells at temperatures higher than 33°C
led to a displacement of the cells within this period, whereas at lower
temperatures no effects of the toxin or forskolin were observed. Following exchange of bathing solutions from N to H, swelling of the
same cells was recorded for a second time (27°C) as described above.
Cells were located by adjusting the coordinates of the motor driven
x-y table of the microscope to the previously
recorded position. This procedure was validated before with fixed and
stained cells.
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Data analysis
To determine the increase of vertical x-z-scan section areas from single cells, the x-z-scan time series were processed using the KS 400 image analyzing software (Carl Zeiss Vision, Hallbergmoos, Germany). A macro-programmed analysis sequence was used, which is schematically shown in Fig. 2. Initially, a single cell was chosen by defining a rectangular region of interest. The subsequent processing included digital smoothing followed by an interactive segmentation of the chosen region to yield a maximally contrasted cell contour, which was thereafter filled with pixels. The number of pixels served as a measure of the vertical x-z-scan section area (a) of a cell. This process was repeated for the entire time series, giving a raw data table of the longitudinal z-section area increase as a function of time, a(t), for each analyzed single cell.
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Calculation of permeability coefficient changes
The osmotic water permeability coefficient Pf can be calculated for single adherent cells by Eq. 1 in a simplified approach explained in detail by Farinas et al. (1995):
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(1) |
represents the time constant of the exponential
function describing cell volume changes due to osmotic challenges, (A/V)o is the ratio of cell
surface area A to volume V before the swelling
reaction, 
o is the initial osmolality of the
bathing solution, and Vw is the molar
volume of water (18 cm3/mol).
The time constant of the swelling process was obtained by fitting
Eq. 2 to the experimentally obtained vertical
x-z-scan section areas a after the
osmotic challenge, assuming that cell shape changes occur only in the
vertical direction (see Results for validation of vertical
x-z-scan section areas as a measure for cell
volume):
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(2) |
determined by the fitting process of the exponential function
a(t) to the raw data of a were
included for further analysis only when the correlation coefficients
R2 for the fitted to the raw data of
a(t) were 
0.90. An example of the obtained fits
to raw data of a swelling IMCD cell before and after stimulation with
forskolin is shown in Fig. 3.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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LSRM and validation of the vertical x-z-scan section area a as a measure for cell volume changes
Fig. 4 shows typical LSRM
x-z-scans of IMCD cells under normotonic
conditions and 8 and 80 s after a challenge with hypotonic bathing
solution, respectively. Although neither fixed nor stained, the cell
shapes are visualized by reflected light in these optical sections. The
figure illustrates that swelling of the cells results in an increase in
cell height, whereas lateral movements are not evident, as indicated by
the unchanged distance between the cells during the swelling process
(bar, 110 µm in all panels). This finding led to the assumption that
the vertical x-z-scan section area of each cell
might serve as an appropriate parameter to monitor cell volume changes
in response to osmotic challenges. To confirm this, experiments were
performed to measure the correlation between vertical
x-z-scan section areas a and the
osmolalities of the bathing solutions. IMCD cells adapted to the cell
growth media (600 mOsm/kg) were challenged with bathing solutions with
osmolalities ranging from 100 to 900 mOsm/kg. After an equilibration
period of 5 min for each osmolality, x-z-scans
were recorded, and vertical x-z-scan section
areas a were subsequently determined for each cell
(n = 5), as described in Materials and Methods and in
Fig. 2. The x-z-scan section areas a
(in percentage of normotonic cells) were then plotted as a function of
bathing solution osmolality (Fig.
5), which could be fitted to a
hyperbolic function for a(), given below in Eq. 6.
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As described in Farinas et al. (1995), cell volume V is
related to solution osmolality by the relationship
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(3) |
x represents the thickness
of the vertical x-z-scan section area
a and s is the vertical section area of osmotically inactive parts assuming the osmolality to be homogenous within (V 
b), then the replacement of the
three-dimensional variables V and b by the
products of vertical areas a with x and of
s with x within Eq. 3 results in
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(4) |
,
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(5) |
s)o with the factor c,
in the relationship
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(6) |
s) to .
Therefore, under the given assumptions, the cell swelling/shrinking
process and thus cell volume changes in response to osmotic challenges
can be delineated from changes of the vertical
x-z-scan section areas a given in Eq. 6. In fact, Eq. 6 could be well fitted to the experimentally obtained
x-z-scan section areas a of IMCD cells
for different osmolalities (Fig. 5) with
R2 > 0.96 (s = 64.81;
c = 22290). This allows the conclusion that the
vertical x-z-scan section area a
serves as a valid parameter to monitor cell volume changes in response
to osmolality changes.
Functional experiments determining osmotic permeability changes
A coverslip with subconfluent IMCD or MDCK cells (see Materials
and Methods) was mounted in a cuvette and transferred to the LSM. LSRM
x-z-scan sections were recorded, while the cells
were challenged osmotically (N to H) by exchanging the bathing
solutions. After a second exchange (H to N), the cells were allowed to
recover from the osmotic hypotonicity challenge on the heating block
(33°C; see Materials and Methods). During this time the IMCD cells
were kept under control conditions (i.e., buffer solution) and were treated with the cAMP-elevating agent forskolin (50 µM, 30 min) or
with toxin B (4 µg/ml; 3 h 30 min), which irreversibly
inactivates small GTPases of the Rho family. MDCK cells, which served
as a negative control for the experiments with toxin B, were much more sensitive to the action of toxin B and were therefore incubated with
lower concentrations of the toxin (40 ng/ml). The effects of C. difficile toxin B on both cell types were assessed before the
functional experiments by fluorescence microscopy observing the
depolymerization of F-actin by TRITC-phalloidin staining (Fig. 6). After this treatment,
hypotonicity-induced cell swelling was again determined using the same
cells. The analysis of the recorded time series for the vertical
x-z-scan section areas a of cells (see
Materials and Methods; Fig. 2) before and after the treatment resulted
in two exponential swelling curves for each cell. Eq. 2 was then fitted
to these raw data curves by nonlinear regression analysis to determine
the time constants of the swelling process, as shown in Fig. 3.
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Investigation of the IMCD cells by transmission and reflection
microscopy (not shown) before and after forskolin treatment revealed
that the cells did not change their shape. Therefore, identical values
for Ao and
Vo were used in Eq. 1. The ratio of time constants before and after forskolin treatment is inversely related to the ratio of permeability coefficients
Pf before and after forskolin
treatment. The reduction of time constants thereby indicates a twofold
increase of the osmotic water permeability coefficient
Pf after forskolin treatment. The
results from time constant determinations before and after different
treatments are shown and summarized in Fig.
7. The values of the time constants before (abscissa) and after (ordinate) treatment, reveal a scattered distribution of data points (each representing data from a single cell)
along a regression line, passing through the origin of the coordinate
system. By virtue of this arrangement of time constants, the
approximate mean change of the water permeability coefficients of the
whole population can be delineated from the inverse slope of the linear
regression line. As expected, IMCD cells undergoing control treatment
(Fig. 7, buffer) show an almost unchanged osmotic water permeability.
Forskolin treatment of IMCD cells, however, induced a doubling of
osmotic water permeability (Fig. 7, forskolin). The attempt to reverse
this acceleration in cell swelling by removal of forskolin from
pretreated cells (four washes with excess of forskolin-free bathing
solution; 15 min each wash) resulted in a reduction of osmotic water
permeability by ~30%, accompanied by a more scattered distribution
of data points around the regression line (Fig. 7, forskolin washout).
This can be explained by a slow and (between cells) variable removal of
forskolin from its intracellular site of action.
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The results show that forskolin increases osmotic water permeability
presumably by elevating cAMP in IMCD cells. The results are in
agreement with the finding that forskolin triggers the translocation of
AQP2-bearing vesicles from intracellular domains into the IMCD cell
membrane (Maric et al., 1998; Klussmann et al., 1999).
We have recently provided evidence for the involvement of small GTPases
of the Rho family in the signal cascade leading to the insertion of
AQP2 into the cell membrane (Klussmann et al., 2001). Application of
toxin B (4 µg/ml; 3 h 30 min) to IMCD cells (see Materials and
Methods) resulted in an acceleration of cell swelling. The inverse
slope of the regression line of the time constants was 2.55 (Fig.
7, toxin B). As toxin B treatment also induced a shape change of IMCD
cells (Klussmann et al., 2001), we included the initial cell surface
Ao and the initial cell volume Vo in the analysis, as implied by Eq. 1. Determinations of Ao and
Vo were performed by a series of
x-y-reflection-mode sections through entire
cells, with a distance of 500 nm between each section (not shown). Cell
surface areas Ao were then calculated
by summarizing the perimeters of each cell slice, multiplied by the
slice thickness, and cell volumes Vo were
calculated by summarizing the areas of each cell slice multiplied by
the slice thickness of 500 nm. Linear regression analysis of the
products of (A/V)o
before and after treatment revealed an inverse slope of 2.048 of the
resulting regression line, thus indicating that toxin B increases the
Pf of IMCD cells to a similar extent
as forskolin (Fig. 7, toxin B corrected for
(A/V)o). The ratio
(A/V)o (3144 ± 346 cm
1) inserted into Eq. 1
allowed the calculation of basal osmotic permeability coefficients of
IMCD cells (Pf = 12.76 ± 3.43 µm/s; n = 12).
To analyze the results of the above experiments statistically, we calculated the quotients of the Pf increases for each individual cell and compared the means of their distributions by nonpaired t-tests (Fig. 7, means of individual cells). There were significant differences between control (buffer-treated) IMCD cells and IMCD cells treated with forskolin (p < 0.0001) and between control and toxin-B-treated cells (p = 0.0132). In contrast, the calculated Pf increases for forskolin and toxin-B-treated IMCD cells did not differ significantly from each other (p = 0.5446). To exclude the possibility that the observed effect of toxin B might have been nonspecific, we also analyzed data obtained with toxin-B-treated MDCK cells. The mean Pf increase observed in MDCK cells after toxin B treatment (40 ng/ml) was not different from buffer-treated IMCD cells (p = 0.4794) but significantly different from forskolin- or toxin-B-treated IMCD cells (p = 0.0454 and p = 0.0223, respectively). Thus, toxin B increases osmotic water permeability in IMCD cells but not in MDCK cells.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We demonstrate here that LSRM is a valid experimental procedure to measure volume changes of adherent cells. As a major advantage of this method, bleaching or quenching effects of fluorophores need not be considered. Other advantages are the acquisition of single-cell kinetic data and the possibility of controlling each cell's swelling behavior by real-time measurements. Cells reacting with excessive, usually irreversible swelling to hypotonic challenge can thus be excluded from further analysis. Laser scanning microscopy is now a broadly established method in cell biology, and the necessary features for the kind of recordings presented here can be relatively easily implemented provided one's instrument is equipped for making true vertical x-z-scans.
To minimize errors we calculated, instead of absolute
Pf values, the changes of
Pf before and after stimulation of the
cells by comparing the time constants of the swelling process. In the case of toxin-B-treated IMCD cells, the increase in
Pf had to be calculated from the
quotient (A/V)o,
because of the shape-changing effect of the toxin.
Pf changes can be delineated quickly
from two-dimensional scatter plots ( values of untreated versus values of the same cells after treatment; Fig. 7).
In the case of primary cultured IMCD cells the osmotic water
permeability was approximately doubled after two different stimuli (forskolin and toxin B). A similar increase was found in
LLC-PK1-cells stably transfected with AQP2
(Katsura et al., 1995). For other cell lines transfected with AQP2,
e.g., CD8 cells (Valenti et al., 1996, 1998) or WT-10 cells (Deen et
al., 1997), a three- to eightfold increase in osmotic water
permeability after forskolin stimulation has been reported using
various experimental approaches. The reason for these differences
between various cell models is not clear, in particular because the
Pf value of unstimulated IMCD cells
(12.76 ± 3.43 µm/s) is in the same range as those reported for
unstimulated CD8 cells and WT-10 cells.
The method presented here can be placed between the one-dimensional,
fluorescent microbeads method and the three-dimensional variant
(preloaded fluorophores). A two-dimensional parameter appears to be
adequate because volume changes of adherent cells reveal basically only
one degree of freedom during cell swelling or shrinkage, namely, that
of cell height, with negligible lateral membrane displacement. Data
reported by Korchev et al. (2000), comparing one- and three-dimensional
approaches to measure osmotically induced cell volume kinetics, as well
as our results indicate the validity of approaches omitting the
calculation of total cell volume. LSRM is a method that leads to valid
results without the requirement for pretreatment of cells with volume
indicators and does not require additional hardware equipment. To our
knowledge, this is the first report introducing reflection microscopy
in combination with laser scanning microscopy in cell biology.
Considered together, the data presented here and those obtained by
Klussmann et al. (2001) suggest that toxin B increases water
permeability in primary cultured IMCD cells by translocation of
AQP2-bearing vesicles from intracellular domains into the cell membrane
in a cAMP-independent manner.
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ACKNOWLEDGMENTS |
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We thank Klaus Aktories (Freiburg, Germany) for providing Clostridium difficile toxin B, John Dickson for critically reading the manuscript, Andrea Geelhaar for superb technical assistance, Ricardo Hermosilla for help in statistical analysis, Brunhilde Oczko for technical assistance in manually analyzing the data sets of the first experimental recordings, Juergen Mevert for manufacturing customized cuvettes for the LSRM experiments, and Robert Storm for performing RT-PCR experiments.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ro 597/6-3) and by a grant from the Fonds der Chemischen Industrie.
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FOOTNOTES |
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Received for publication 9 August 2000 and in final form 2 January 2001.
Address reprint requests to Dr. Kenan Maric, Forschungsinstitut für Molekulare Pharmakologie, Robert-Roessle-Strasse 10, 13125 Berlin, Germany. Tel.: 49-030-94793-260; Fax.: 49-030-94793-109; E-mail: maric{at}fmp-berlin.de.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Biophys J, April 2001, p. 1783-1790, Vol. 80, No. 4
© 2001 by the Biophysical Society 0006-3495/01/04/1783/08 $2.00
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 R. Storm, E. Klussmann, A. Geelhaar, W. Rosenthal, and K. Maric Osmolality and solute composition are strong regulators of AQP2 expression in renal principal cells Am J Physiol Renal Physiol, January 1, 2003; 284(1): F189 - F198. [Abstract] [Full Text] [PDF]
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E. Klussmann, G. Tamma, D. Lorenz, B. Wiesner, K. Maric, F. Hofmann, K. Aktories, G. Valenti, and W. Rosenthal An Inhibitory Role of Rho in the Vasopressin-mediated Translocation of Aquaporin-2 into Cell Membranes of Renal Principal Cells J. Biol. Chem., June 1, 2001; 276(23): 20451 - 20457. [Abstract] [Full Text] [PDF]
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G. Tamma, E. Klussmann, K. Maric, K. Aktories, M. Svelto, W. Rosenthal, and G. Valenti Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells Am J Physiol Renal Physiol, December 1, 2001; 281(6): F1092 - F1101. [Abstract] [Full Text] [PDF]
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