§
From the * Departamento de Neurobiología, Instituto Mexicano de Psiquiatría, México 14370, D.F. México; the Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555; and the § Departamento de Farmacología y Toxicología, Centro de Investigación y de Estudios Avanzados del IPN, México 07000, D.F. México
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ABSTRACT |
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The possible role of Ca2+ as a second messenger mediating regulatory volume decrease (RVD) in osmotically swollen cells was investigated in murine neural cell lines (N1E-115 and NG108-15) by means of novel microspectrofluorimetric techniques that allow simultaneous measurement of changes in cell water volume and
[Ca2+]i in single cells loaded with fura-2. [Ca2+]i was measured ratiometrically, whereas the volume change was determined at the intracellular isosbestic wavelength (358 nm). Independent volume measurements were done using calcein, a fluorescent probe insensitive to intracellular ions. When challenged with ~40% hyposmotic solutions, the cells expanded osmometrically and then underwent RVD. Concomitant with the volume response, there was a transient increase in [Ca2+]i, whose onset preceded RVD. For hyposmotic solutions (up to ~40%), [Ca2+]i
increased steeply with the reciprocal of the external osmotic pressure and with the cell volume. Chelation of external and internal Ca2+, with EGTA and 1,2-bis-(o -aminophenoxy) ethane-N,N,N ',N '-tetraacetic acid (BAPTA), respectively, attenuated but did not prevent RVD. This Ca2+-independent RVD proceeded even when there was a
concomitant decrease in [Ca2+]i below resting levels. Similar results were obtained in cells loaded with calcein.
For cells not treated with BAPTA, restoration of external Ca2+ during the relaxation of RVD elicited by Ca2+-free
hyposmotic solutions produced an increase in [Ca2+]i without affecting the rate or extent of the responses. RVD and the increase in [Ca2+]i were blocked or attenuated upon the second of two ~40% hyposmotic challenges applied at an interval of 30-60 min. The inactivation persisted in Ca2+-free solutions. Hence, our simultaneous measurements of intracellular Ca2+ and volume in single neuroblastoma cells directly demonstrate that an increase in
intracellular Ca2+ is not necessary for triggering RVD or its inactivation. The attenuation of RVD after Ca2+ chelation could occur through secondary effects or could indicate that Ca2+ is required for optimal RVD responses.
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INTRODUCTION |
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Neurons, like all animal cells, possess mechanisms for
maintaining constant volume in isosmotic media in the
face of the Donnan effect or changes in intracellular
solute content, such as those produced by neurotransmitters, hormones, repetitive nerve impulses, or upon
sodium pump inhibition (Serve et al., 1988; Alvarez-Leefmans et al., 1992
). In addition, neurons, like most
cells, regulate their volume when exposed to anisosmotic media (Hoffmann and Simonsen, 1989
). Upon
exposure to hyposmotic media, cells initially swell, and
then return to their initial volume, a phenomenon
termed regulatory volume decrease (RVD).1 This regulatory response is accomplished through the loss of intracellular solutes along with osmotically obligated water. Three components are involved in this negative
feedback loop: (a) the volume sensor, which detects a
change in cell volume and transduces this change into
an intracellular signal; (b) the effector mechanism that
causes the loss of osmolytes and water, thereby correcting the aberrant volume; and (c) the signal or second
messenger that couples the sensor to the effector. Chief
among the candidates for second messenger is intracellular Ca2+ (for review see McCarty and O'Neil, 1992
).
However, the evidence supporting a signaling role for
Ca2+ is fragmentary and controversial (Grinstein and
Smith, 1990
; Foskett, 1994
). For instance, RVD measured in cell populations of neurons or astrocytes has
been claimed to be unaffected by external Ca2+ removal,
although internal Ca2+ was neither chelated nor measured (Pasantes-Morales et al., 1993
, 1994
). In neuroblastoma cells, RVD occurred in the absence of external
Ca2+ in cells loaded with the Ca2+ chelator 1,2-bis-(o -aminophenoxy) ethane-N,N,N ',N '-tetraacetic acid (BAPTA;
Lippmann et. al., 1995). However, intracellular Ca2+
was not measured to verify the effectiveness of Ca2+
buffering. A role for Ca2+ as mediator of RVD has been
claimed for various cell types (McCarty and O'Neil,
1992
) including astrocytes (O'Connor and Kimelberg, 1993
) and cortical neurons (Churchwell et al., 1996
).
More recently, it was concluded that Ca2+ signaling by
Ca2+ release or Ca2+ entry appears to play no role in
the activation mechanism for the RVD responses in
Ehrlich cells (Jørgensen et al., 1997
). However, changes
in intracellular free Ca2+ concentration, [Ca2+]i, were
measured in cell populations or on single cells, in
which changes in volume were not measured simultaneously. Clearly, part of the above controversy is due to
the fact that changes in [Ca2+]i and volume have not
been measured simultaneously in single cells and a
causal relationship between these two variables has not
been definitively established in the above studies.
In the present study, we unify the above conflicting
claims in the literature by directly demonstrating that
RVD can proceed independently of a rise in intracellular Ca2+, but that there is a component of the response
that is sensitive to external and internal Ca2+ chelation.
We used novel optical techniques to simultaneously measure changes in [Ca2+]i and cell volume in response to hyposmotic challenges in two neuronal cell
lines (N1E-115 and NG108-15). Changes in cell water volume (CWV) were estimated from the change in intracellular concentration of trapped fluorescent dyes
(Alvarez-Leefmans et al., 1995). By using the Ca2+-sensitive dye fura-2 and recording at the Ca2+-sensitive
(380 nm) and -insensitive (isosbestic, 358 nm) wavelengths, we monitored simultaneous changes in CWV
and [Ca2+]i in single cells (Muallem et al., 1992
). CWV
changes were also measured using calcein as the fluorescent probe (Crowe et al., 1995
), thus providing independent measurements of CWV with a dye having different chemical properties than fura-2.
We find that upon hyposmotic swelling there is a rise
in [Ca2+]i whose onset precedes RVD. However, chelation of external and internal Ca2+ only attenuates, but
does not prevent, RVD. Moreover, this Ca2+-independent RVD proceeds even when [Ca2+]i decreases below
resting level. We also find that upon multiple hyposmotic challenges the RVD response is lost. We call this
effect inactivation. This phenomenon is also independent of Ca2+. Two models are consistent with our observations. In the first model, a single sequential process underlies RVD. In this model, a rise in [Ca2+]i cannot be the triggering signal for RVD. The attenuation
of RVD after Ca2+ chelation could occur through secondary effects or could indicate that Ca2+ is required
for optimal responses. In the second model, RVD could be the consequence of the operation of two parallel
processes, one of which is Ca2+ independent. Some of
these results have been reported in preliminary form
(Altamirano et al., 1996).
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MATERIALS AND METHODS |
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Cell Culture
Two murine neural cell lines maintained in culture, N1E-115
neuroblastoma and neuroblastoma × glioma NG108-15, were
used in the present experiments. N1E-115 is a clone of cells derived from mouse neuroblastoma C-1300 (Amano et al., 1972),
and expresses characteristics of neuronal cells. The neuroblastoma × glioma hybrid cells NG108-15 are hybrid cells obtained
from a rat glioma cell line and a mouse neuroblastoma line, and express characteristics of both glial and neuronal cells (Hamprecht,
1977
). Cells were grown at 37°C on culture dishes containing
90% Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 1% hypoxanthine-aminopterin-thymidine (HAT) and 1% L-glutamine, in a 5% CO2/95% air atmosphere.
Cells from passages 9-25 were plated on 25-mm-diameter glass
coverslips (Bellco Glass, Inc., Vineland, NJ), previously treated
with poly-D-lysine. Differentiation was induced 24 h after plating
by supplying the cells with a low serum growth medium composed of 98% DMEM, 1% FCS, 1% HAT, 1% L-glutamine, 1 mM
theophylline, and 10 µM prostaglandin E1 (Kasai and Neher,
1992
). Cells were used from 1 to 14 d after the differentiation
treatment.
Saline Solutions
The standard external solution (SES) contained (mM): 130 NaCl, 5.5 KCl, 2.5 CaCl2, 1.25 MgCl2, 20 HEPES, 10 dextrose, 13 sucrose. The pH was adjusted to 7.3 with NaOH and the osmolality was 312 ± 3 mosmol/kg water. The control isosmotic solution was prepared by substituting 65 mM NaCl for sucrose to match the osmolality of the culture medium (312 mosmol/kg water). Anisosmotic solutions were prepared by sucrose addition or removal to get the desired osmolality, thus maintaining constant the ionic strength and at the same value as the isosmotic control. Anisosmotic solutions were expressed as the decrement (or increment) percentage with respect to the control isosmotic solutions. Thus, a solution referred to as a "44% hyposmotic" means that its measured osmolality was 56% of the control isosmotic solution. The zero Ca2+ solutions were made by substituting CaCl2 for MgCl2 and adding 0.5 mM EGTA. The osmolality of all solutions was measured with a vapor pressure osmometer (5100 B; Wescor Inc., Logan, UT).
Dye Loading and Bath Chamber
Each coverslip with the cells attached was mounted in a Leiden
chamber (Medical Systems Corp., Greenvale, NY) filled with SES
and placed on the stage of an epifluorescence inverted microscope. Cells were loaded with the fluorophores fura-2 or calcein
as described previously (Alvarez-Leefmans et al., 1995). In brief,
they were incubated at room temperature (22°C) in SES containing either 5 µM fura-2 acetoxymethyl ester (AM), or 2 µM calcein/AM. To prepare the fura-2-loading solution, we used a stock
containing 50 µg fura 2/AM dissolved in 4 µl DMSO plus 4 µl
pluronic (10% wt/wt in DMSO). The final concentration of fura-2/AM for this stock solution was 6.23 mM. The calcein loading
solution was prepared from a stock containing 50 µg calcein/AM
dissolved in 4 µl DMSO plus 4 µl pluronic (10% wt/wt in
DMSO). The final calcein/AM concentration of this stock solution was 6.28 mM. Dye loading was monitored fluorometrically by
sampling a single cell every 30 or 60 s until reaching the desired
level of fluorescence. For calcein, the level was
14.5× the initial
fluorescence (i.e., the cell fluorescence without dye), and for
fura-2 the level was
8, at 358 nm. The loading time was 37 ± 3 min for calcein (n = 22) and 43 ± 3 min for fura-2 (n = 26). The
loading solution was then washed out with SES for at least 1 h before starting the experimental measurements. All solutions, except those used for loading (see below), were perfused into the
chamber at a rate of 3 ml/min. The chamber fluid was exchanged
with a time constant of 3.6 ± 0.3 s.
BAPTA Loading
To buffer intracellular Ca2+ near resting levels, in some experiments we used the Ca2+ chelator BAPTA. In this series of experiments, after the cells were loaded with either calcein or fura-2 as described above, they were briefly washed with SES and loaded with BAPTA. This was accomplished by incubating the cells at room temperature for 2 h in SES containing 100 µM BAPTA-AM. The BAPTA loading solution was prepared from a stock containing 3.82 mg of BAPTA/AM dissolved in 1 ml DMSO. After BAPTA loading, cells were superfused with SES for 1 h before starting the experimental measurements. The chelating action of intracellular BAPTA was verified by ratiometric measurements with fura-2.
Measurement of Cell Volume with Fluorescent Dyes
Basic principle.
Changes in CWV can be assessed by measuring
changes in concentration of impermeant substances (volume
markers) introduced into cells (Alvarez-Leefmans et al., 1995).
Intracellularly trapped fluorescent dyes such as calcein (Crowe et
al., 1995
) and fura-2 (Muallem et al., 1992
) have been successfully used as volume indicators. These optical techniques provide
superior time resolution and sensitivity relative to other techniques and, when using ratiometric dyes, they allow parallel measurements of changes in CWV and intracellular ion concentrations in single cells. Changes in CWV are estimated from changes
in fluorescence intensity of the dye resulting in turn from
changes in its intracellular concentration. By using the Ca2+-sensitive dye fura-2 and recording at the Ca2+-sensitive (380 nm) and
-insensitive (isosbestic) wavelengths, we recorded simultaneously
changes in CWV and [Ca2+]i in single cells. CWV changes were
inferred from changes in the concentration of fura-2, recorded at
the isosbestic wavelength (see below). The simultaneous changes
in [Ca2+]i were monitored by taking the ratio 358/380. Independent and parallel measurements of CWV were provided by using
calcein, a dye that is more intensely fluorescent than fura-2 and
insensitive to changes in the concentration of native cellular ions
within physiological ranges. The validation, pitfalls, and limitations of these techniques have been discussed in detail in previous publications (Alvarez-Leefmans et al., 1995
; Crowe et al.,
1995
, 1996
).
Fluorescence measurements.
Total fluorescence from a small region of fluorophore-loaded single cells was measured with a customized microspectrophotometry system that has been described
in detail elsewhere (Alvarez-Leefmans et al., 1995). The system
included an inverted, epi-fluorescence microscope (Diaphot-TMD; Nikon, Tokyo, Japan) equipped with a fluor oil-immersion
objective lens (40×, 1.3 NA; Nikon). The excitation light coming
from a 150 W xenon arc lamp passed through a water filter, and
was then divided by a beam splitter. Each beam passed through a
computer controlled high speed shutter (Uniblitz; Vincent Assoc., Rochester, NY). Attached to each shutter was a filter wheel
holding a selection of customized excitation filters (Omega Optical, Brattleboro, VT). For fura-2 measurements, beam 1 passed through a filter centered at 380 ± 6 nm. For exciting fura-2 at its
isosbestic wavelength, beam 2 passed through a filter centered at
362 or 358 ± 5 nm. The 358-nm wavelength was preferred over the 362-nm because it was closer to the intracellular isosbestic wavelength measured in vivo with a monochromator and the
same objective lens used in the present work (Crowe et al., 1996
). In some experiments using the monochromator, measurements
were done at wavelengths of 350 and 358 nm (slit = 8 nm) to optimize the sampling rate.
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Calculation of cell water volume changes.
Normalized cell water
volume changes (Vt/Vo) were computed from monitored changes
in relative fluorescence (Ft/Fo) resulting from exciting fura-2 at
358 nm or calcein at 495 nm, according to the following equation
(Alvarez-Leefmans et al., 1995):
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(1) |
Drift Correction and Statistical Analysis
When necessary, the drift of the fluorescence signal resulting from dye leakage and photobleaching was corrected by fitting a linear regression to the base line and multiplying the slope of this regression line by the time at which each data point was sampled. This process yields point by point drift values that, in turn, are subtracted from the record yielding the corrected trace.
RVD response variables were measured as defined in Fig. 2. The extent of RVD (Fig. 2 f ) was measured 20 min after the onset of the regulatory response and is presented as
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(2) |
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where peak Vt/Vo and reg Vo/Vt are the peak and regulated relative volumes, respectively. Therefore,% RVD denotes the magnitude of the return from the peak swollen volume back toward the
isosmotic initial volume, Vo, such that 0% RVD indicates no volume regulation and 100% RVD indicates complete volume recovery. Referring to Fig. 2, % RVD is simply [(a g)/a] × 100. All
measurements are expressed as mean ± SEM. The one-tailed unpaired t test was used to establish the significance of differences
between means at the 95% confidence limit.
Chemicals
All chemicals used to prepare bathing media came from Sigma Chemical Co. (St. Louis, MO). Tissue culture media (Dulbecco's modified Eagle's medium), L-glutamine, and hypoxanthine-aminopterin-thymidine came from GIBCO BRL (Gaithersburg, MD), FCS from Hyclone (Logan, UT), theophylline and prostaglandin E1 from Sigma Chemical Co., and poly-D-lysine from ICN (Costa Mesa, CA). Fura-2/AM, fura-2-free acid, calcein/AM, BAPTA/AM pluronic, and calcium buffers came from Molecular Probes, Inc. (Eugene, OR).
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RESULTS |
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Regulatory Volume Decrease in Single Cells Loaded with Calcein or Fura-2
Regulatory volume decrease was studied in cultured N1E-115 and NG108-15 cells loaded with either calcein or fura-2. This strategy provided an independent measurement of CWV with dyes having different chemical properties, thus giving more reliability to our observations. In particular, calcein fluorescence is independent of changes in the concentration of native intracellular ions within the physiological range. The parallel use of calcein eliminates possible intracellular Ca2+ buffering effects of fura-2, which may attenuate putative Ca2+-dependent components of the RVD response pattern. On the other hand, the use of fura-2 allowed for the simultaneous measurement of changes in [Ca2+]i and CWV in single cells.
Fig. 3 shows a typical RVD pattern in response to a
hyposmotic challenge recorded in a single NG108-15
cell loaded with calcein. The cell was initially superfused with an isosmotic solution to obtain a stable baseline. Upon exposure to a 44% hyposmotic solution, the
calcein relative fluorescence (Ft/Fo) decreased due to
intracellular dye dilution resulting from net osmotic influx of water. After reaching a minimum, the Ft/Fo signal recovered close to initial values, reflecting a gradual
increase in intracellular dye concentration resulting
from regulatory net water efflux. When the hyposmotic solution was replaced with the isosmotic solution, the
Ft/Fo signal increased beyond control levels, reached a
peak, and recovered to initial values. This last sequence
of fluorescence signals was also expected because,
upon returning to the isosmotic solution from a hyposmotic one, the isosmotic solution is hypertonic with respect to the cell that shrinks, and then recovers its initial water volume. Fluorescence signals like these were
transformed into relative cell water volume changes
(Vt/Vo) using Eq. 1. This transformation yielded traces
like the one shown in the bottom of Fig. 3. Before testing the 44% hyposmotic solution, the cell was exposed
to calibration test solutions (Fig. 3, inset). These calibration solutions had osmolalities of +10 and 12% with
respect to the isosmotic solution. The inset includes a
plot that was routinely made for each cell, showing the
relationship between steady state values of changes in
Vt/Vo and the reciprocal of the relative osmotic pressure of the medium (
o/
t). The line denotes the predicted behavior of a perfect osmometer according to
the equation:
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(3) |
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where Vt, Vo, o, and
t, have been defined. (Fig. 3,
)
Osmotic calibration pulses. Note that the measurements fall close to the theoretical line for a perfect osmometer. (Fig. 3,
) The peak amplitude of the osmotic response produced by the 44% hyposmotic solution, which also falls close to the theoretical line
denoting ideal behavior, before RVD ensues.
Experiments like the one described were performed
in seven NG108-15 cells. Upon exposure to a solution
~40% hyposmotic (41.3 ± 1%), the cells swelled to a
maximum of 69 ± 5% above their initial volume, which
is not significantly different from the value of 71 ± 3%
predicted for ideal behavior. The time to reach the
maximum swelling was 4.4 ± 0.5 min. After a delay of
47 ± 6 s measured from the peak of the osmotic swelling, RVD ensued at an average initial rate of 3.4 ± 0.6% min
1 with a partial volume recovery at 20 min
(percent RVD recovery) of 57 ± 11%. Upon returning
to the control isosmotic solution (which would now be
hypertonic with respect to the cells), the cells shrank
and their volume started to recover. In two cells, complete recovery occurred at 37 and 103 min, respectively,
upon returning to the isosmotic solution. The above results are summarized in Table I. Similar results were obtained in three N1E-115 neuroblastoma cells (see Crowe
et al., 1995
).
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The prevailing controversy about the possible role of
intracellular Ca2+ as the signal that couples the activation of the volume sensor and the transport mechanisms for RVD prompted us to measure simultaneously [Ca2+]i and volume changes in fura-2-loaded cells. Fig.
4 shows that a 42% hyposmotic solution produced an
increase in [Ca2+]i accompanied by RVD. When exposed to an ~40% hyposmotic solution (41.4 ± 0.6%),
fura-2-loaded NG108-15 cells (n = 10) swelled to a maximum of 59 ± 5% above their initial volume. The time
to reach the maximum swelling was 5.0 ± 0.4 min. After a delay of 90 ± 23 s, RVD ensued at an average initial rate of 3.0 ± 0.5% min
1 with partial recovery
(percent RVD) of 45 ± 8%. The ratio 358/380, which
signals changes in [Ca2+]i, started to increase with a latency of 29.5 ± 12 s measured from the onset of the volume change produced by the hyposmotic challenge.
The ratio peaked at 7.6 ± 1.4 min after the onset of the
volume change produced by the hyposmotic challenge;
i.e., ~1 min after the onset of RVD. Similar results were
obtained in N1E-115 cells (n = 4) following the same
protocol. However, in cells loaded with fura-2, the
mean values of some variables of RVD responses with
respect to those recorded in calcein-loaded cells were
slightly attenuated (e.g., maximum swelling and percentage of RVD) or slowed down (e.g., time to peak
swelling), but the differences were not statistically significant (Table I).
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These results show that fura-2-loaded cells behave like those loaded with calcein in terms of their response to anisosmotic solutions and that the change in cell volume is parallel to a rise in [Ca2+]i. Moreover, the temporal relationship between changes in [Ca2+]i and RVD is appropriate for a putative signaling role of internal Ca2+. However, the results do not prove a causal relation between the rise in [Ca2+]i and RVD.
Relationship between External Osmolality, [Ca2+]i, and Cell Volume
To test further the Ca2+ hypothesis for RVD, we recorded simultaneously changes in volume and [Ca2+]i
in cells loaded with fura-2 that were exposed to solutions of various osmolalities. Fig. 5 A shows an example
in an NG108-15 cell. No measurable changes in the
[Ca2+]i signal were produced by a 14% hyperosmotic
solution that caused an osmometric shrinkage. For
hyposmotic solutions, [Ca2+]i increased steeply with
o/
t (Fig. 5 B,
) and cell volume (Fig. 5 C ). The
10% hyposmotic solution resulted in an osmometric increase in cell volume accompanied by a modest rise in
[Ca2+]i (Fig. 5 A). There was no sign of RVD response.
The 26 and 40% hyposmotic solutions resulted in respective transient increases in [Ca2+]i as a function of
o/
t and Vt/Vo, and clear RVD responses in Fig. 5 A, I
and II . The rate of RVD increased with [Ca2+]i and Vt/Vo.
The peak of the responses I and II fall close to the value
predicted for the behavior of a perfect osmometer (Fig. 5 B, I and II ), thus confirming that RVD started after
the cell had increased its water volume in an osmometric manner. The above findings show again a close parallel between changes in [Ca2+]i, cell volume, and rate
of RVD. However, they do not prove that the increase
in [Ca2+]i upon osmotic swelling is a necessary causal
signal for RVD and not just an independent parallel
process.
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Changes in [Ca2+]i and RVD upon Repeated Hyposmotic Challenges
The close parallel between changes in [Ca2+]i and RVD was further shown by exposing the cells to two 40% hyposmotic challenges, each lasting ~40 min, applied with an interval of 30-60 min. RVD and the concomitant increase in [Ca2+]i did not occur or were significantly attenuated upon exposure to the second hyposmotic challenge. Fig. 6 illustrates one of these experiments performed in an N1E-115 cell. The first hyposmotic challenge produced the expected transient increase in [Ca2+]i accompanied by RVD. However, the second hyposmotic challenge, applied 55 min after the end of the first one, produced a measurable increase in neither [Ca2+]i nor RVD. In some experiments, the second hyposmotic challenge produced an increase in [Ca2+]i, albeit smaller than the one evoked by the first hyposmotic challenge, without a concomitant RVD response. The disappearance of the RVD response upon the second hyposmotic shock was also observed in calcein-loaded cells in both cell lines (Fig. 7).
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To test for the external Ca2+ dependence of RVD inactivation, calcein-loaded cells were exposed to Ca2+-free isosmotic and hyposmotic solutions containing 0.5 mM EGTA. The cells were first superfused with the Ca2+-free isosmotic solution for at least 10 min, and then the Ca2+-free 40% hyposmotic solution was applied twice with an interval of ~50 min. The results show that both RVD and its inactivation upon the second hyposmotic challenge proceeded in the virtual absence of external Ca2+ (Fig. 7).
We conclude that RVD inactivation is independent of (a) the type of dye with which the cells are loaded, (b) the cell type (NG108-15 or N1E-115), and (c) the presence of external Ca2+.
The fact that RVD disappearance coincides with suppression or attenuation of the concomitant change in [Ca2+]i confirms the parallel between a rise in [Ca2+]i and RVD.
Effect of Extracellular and Intracellular Ca2+ Chelation on RVD and [Ca2+]i
The experiments so far described demonstrate a tight
correlation between changes in [Ca2+]i and RVD. Demonstration of this correlation is necessary but not sufficient to suggest a causal role for Ca2+ as a second messenger mediating RVD. A crucial test for the Ca2+ hypothesis is to show directly that RVD is abolished when
the rise in intracellular Ca2+ upon osmotic swelling is
inhibited. Elevations of [Ca2+]i produced by hyposmotic solutions could result either from Ca2+ influx
across the plasma membrane or Ca2+ release from internal stores (McCarty and O'Neil, 1992; Wu et al.,
1997
). Thus, we studied the effect of extracellular and
intracellular Ca2+ chelation on RVD. To remove the external source of Ca2+, these experiments were done
with Ca2+-free EGTA isosmotic and hyposmotic solutions. Intracellular Ca2+ was chelated with BAPTA. For
this purpose, cells were incubated with 100 µM
BAPTA/AM for 2 h, loaded with calcein, and then superfused with the Ca2+-free EGTA isosmotic solution
for at least 10 min before the Ca2+-free EGTA hyposmotic solution. The use of calcein was important in these
experiments because this dye does not affect intracellular Ca2+ and does not have the side effects of fura-2. We
compared a control population of cells with those
treated with BAPTA and Ca2+-free EGTA solutions. Such
Ca2+ chelation did not abolish RVD; however, there was
a significant (P < 0.01) decrease in the rate (53%) and
extent (56%) of RVD responses (Table II).
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The persistent RVD could be due to incomplete intracellular Ca2+ chelation. The ability of BAPTA to chelate intracellular Ca2+, like that of other Ca2+ chelators, depends on the [Ca2+]i, the ionic strength and composition of the cytosol, and the [BAPTA]i. Unless one knows these factors, it is not possible to be sure that BAPTA is adequately chelating internal Ca2+. In the absence of such knowledge, it is essential to verify that BAPTA is adequately chelating intracellular Ca2+. To verify that BAPTA was actually damping any hyposmotically induced increases in [Ca2+]i, another series of experiments was done with fura-2-loaded cells. Under these conditions, the hyposmotic challenge resulted in a decrease in [Ca2+]i below resting levels, but the RVD response persisted (Fig. 8).
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In seven fura-2-loaded cells treated following the
above protocol, upon exposure to the Ca2+-free ~40%
hyposmotic (42.6 ± 0.4%) solution, the cells swelled to
a maximum of 62 ± 4% above their initial volume. The
time to reach the maximum swelling was 4.9 ± 0.5 min.
After a delay of 54 ± 8 s, RVD ensued at an initial average rate of 1.6 ± 0.5% min
1 with 30 ± 6% partial recovery (percent RVD). The mean values of all these
variables were smaller than those measured in fura-2-loaded control cells without BAPTA, but the differences were statistically significant only for the initial
rate of RVD (Table III and Fig. 9 A). However, the differences in the average rate and extent of RVD in the
Ca2+-free EGTA-BAPTA-treated cells loaded with fura-2
compared with the calcein-loaded control cells were
statistically significant (P < 0.05). The Ca2+ chelation
effects on the extent of RVD responses were not statistically significant when compared with the control cells
loaded with fura-2 (Table III), probably because the latter indicator was already exerting some chelating or
other side effects in the control group. In other words,
the control responses in the fura-2-loaded cells were
slightly blunted from the beginning. This can be appreciated by comparing the "control" parameters between
calcein-loaded and fura-2-loaded cells (Table I and Fig.
9). Again, although the differences between controls are
not statistically significant, the tendencies are obvious.
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These results prove that RVD can be produced in the absence of a rise in internal Ca2+, suggesting that if a single sequential process underlies RVD, a rise in [Ca2+]i cannot be the triggering signal. The effects of Ca2+ removal suggest that Ca2+ may be acting as a modulator affecting RVD kinetics, or chelation may affect RVD through secondary mechanisms.
The Rate of RVD Responses Elicited with Ca2+-free Hyposmotic Challenges Is Not Affected by Restoring External Ca2+
An increase in [Ca2+]i in isosmotic media produces
shrinkage of N1E-115 cells (Crowe et al., 1995). This
suggests that these cells are endowed with Ca2+-sensitive mechanisms for net solute efflux, which can be activated by Ca2+ in isosmotic media. The question arises
as to whether such Ca2+-triggered mechanisms can be
activated during osmotic swelling. To examine this
question, experiments were done in which [Ca2+]i was
increased during ensuing RVD responses elicited by exposure to a Ca2+-free EGTA hyposmotic solution. The
rationale was that if Ca2+-dependent processes are important, then the rate of cell volume recovery would be
enhanced by an increase in [Ca2+]i. In these experiments, we took advantage of the fact that after superfusion with a Ca2+-free solution, restoration of [Ca2+]o results in an increase in [Ca2+]i. Fig. 10 A shows one such
experiment in a fura-2-loaded cell. Note that upon removal of external Ca2+ in the isosmotic solution, the
basal level of [Ca2+]i decreased. When a Ca2+-free 40%
hyposmotic solution was applied, the relative cell volume increased and the cell started to downregulate its
volume. A Ca2+-containing hyposmotic solution was
then applied while the cell was still undergoing RVD.
This resulted in the expected transient increase in
[Ca2+]i. However, the rate of RVD (Fig. 10 A, top) did
not increase. Similar experiments were conducted in
calcein-loaded cells (n = 7). An example is shown in
Fig. 10 B in which readmission of external Ca2+ during
an RVD response elicited by exposure to a 40% Ca2+-free hyposmotic solution did not alter its time course.
|
The results obtained in six calcein-loaded cells are summarized in Fig. 11. External Ca2+ was readmitted in a time window between 8 and 14 min after RVD ensued. The time at which external Ca2+ was readmitted in each experiment is indicated by the large gray circles. The filled circles correspond to mean values ± SEM of the six RVD responses. No sign of enhancement of RVD rate upon external Ca2+ readmission was observed.
|
Other experiments were done (n = 3) in which [Ca2+]i was increased during osmotic swelling by adding 1 µM ionomycin in fura-2-loaded cells. The results were similar; i.e., no significant enhancement of RVD rate could be demonstrated. On the basis of these results, we suggest that Ca2+ is not a triggering signal for RVD in osmotically swollen cells, at least within the explored time window.
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DISCUSSION |
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Calcium-dependent and Calcium-independent RVD
The nature of the signal transducing the change in cell
volume to the activation of osmolyte efflux pathways for
RVD in osmotically swollen cells is not clear. Two major
models have been proposed, one involving Ca2+ as an
intracellular second messenger and the other in which
RVD is mediated through Ca2+-independent mechanisms. According to the first model, cell swelling causes
an increase in cytosolic Ca2+ that activates volume regulatory mechanisms. The increase in [Ca2+]i may result
from Ca2+ influx, via plasma membrane channels
(Christensen, 1987), or Ca2+ release from intracellular
stores (McCarty and O'Neil, 1992
; Wu et al., 1997
). The
proposed effectors are mostly Ca2+-activated cation and
anion channels and carriers. This model has been
widely accepted as a paradigm for the involvement of Ca2+ as a transducing signal for RVD. Nevertheless, the
existing evidence backing up this model is often weak
and fragmentary, and the comprehensive analysis necessary to establish an active role of cytosolic Ca2+ for
RVD is lacking in many of the cells in which it has been implicated (Foskett, 1994
). One of the experimental
facts that is missing in the work claiming a signaling
role for Ca2+ in RVD is the simultaneous measurement
of changes in [Ca2+]i and cell volume, and the demonstration that the Ca2+ signal is the cause of RVD and
not just a parallel phenomenon. In the second model,
RVD is effected by Ca2+-independent mechanisms (Cahalan and Lewis, 1988
; Grinstein and Smith, 1990
; Foskett et al., 1994
; Lippmann et al., 1995
). According to
this model, hyposmotic swelling opens anion channels,
causing Cl
efflux with consequent cell membrane depolarization towards the equilibrium potential for Cl
.
This depolarization activates voltage-gated K+ channels
and increases the driving force for K+ loss. The end result is the electrically coupled exit of K+ and Cl
, accompanied by water. The volume-activated anion channels may be sufficiently nonselective to permit efflux of
organic anions, as well as inorganic anions such as Cl
and HCO3
(Strange et al., 1996
). The mechanism
whereby these anion channels are activated upon cell
swelling remains to be elucidated. However, with the
exception of a recent study in human neuroblastoma
cells (Basavappa et al., 1995
), there is general agreement that Ca2+ is not the activating signal of these anion channels (Doroshenko and Neher, 1992
; Strange et
al., 1996
). In addition to channel-mediated solute efflux, there are carrier-mediated efflux pathways such as
electroneutral KCl cotransport that can be activated in
swollen cells in the virtual absence of Ca2+ (Thornhill
and Laris, 1984
; Kramhøft et al., 1986
).
Unlike previous reports, in the present study we measured changes in CWV and [Ca2+]i simultaneously in
the same cells. We found that in single N1E-115 neuroblastoma cells and in NG108-15 hybrid neuroblastoma × glioma cells, there is a close parallel between osmotic
swelling, increase in [Ca2+]i, and RVD. The extent and
rate of rise of [Ca2+]i and RVD were proportional to
the degree of medium hypotonicity and the change in
cell volume. Moreover, the correlation between the increase in cell volume, the initiation of the rise of
[Ca2+]i, and subsequent [Ca2+]i changes, and the onset
of RVD show the appropriate time sequence to suggest
that a rise in [Ca2+]i might, in principle, mediate RVD.
However, cells bathed in Ca2+-free EGTA solutions,
loaded with both the Ca2+ chelator BAPTA and the
Ca2+ indicator fura-2, showed a robust RVD without a
concomitant rise in [Ca2+]i. Moreover, the persistent
RVD occurred even when [Ca2+]i decreased below resting levels. Thus, no cause-effect relation exists between
the rise in intracellular Ca2+ and the RVD response.
These results directly prove for the first time that if a
single process underlies RVD, a rise in [Ca2+]i is not
the triggering signal. The notion that Ca2+ plays no
role in the activation of the normal RVD response has been suggested by other investigators based on less direct evidence from experiments on cell populations in
which no simultaneous measurements of intracellular
Ca2+ and volume were made (e.g., Jørgensen et al.,
1997). Cell populations may or may not be homogeneous and may or may not respond synchronously to
osmotic challenges. Therefore, experiments in cell
populations do not allow reliable inferences and definitive conclusions about what is happening in single cells,
particularly when the relevant variables (changes in cell
volume and [Ca2+]i) are not recorded simultaneously.
The rate and extent of the RVD responses that persisted after Ca2+ chelation were reduced with respect to
control responses. External and internal Ca2+ chelation may attenuate the RVD response through secondary effects; e.g., membrane depolarization leading to a
decreased driving force for Cl efflux or side effects of
BAPTA such as intracellular acidification or loss of K+
and Cl
(Jørgensen et al., 1997
). Alternatively, the results could be the consequence of the operation of two
parallel processes activated by osmotic swelling, one of
which is Ca2+ independent. In this model, the rate and
extent of RVD would be affected by Ca2+ chelation simply by the elimination of the Ca2+-sensitive component.
The role of Ca2+ in the latter component is not clear at
this time. It could be that a certain resting level of intracellular (and extracellular) Ca2+ may be necessary for optimal RVD responses, as has been suggested for a medullary thick ascending limb cell line derived from rabbit kidney (Montrose-Rafizadeh and Guggino, 1991
).
According to this view, Ca2+ might play a permissive
role, for example acting as a cofactor or maintaining
cell integrity, rather than acting as a second messenger.
Ca2+ Regulatory Mechanisms in Anisosmotic and Isosmotic Media
Our conclusion for a Ca2+-independent RVD is
strengthened by the fact that the rate of cell volume recovery was not enhanced by restoring external Ca2+
within an interval of 8-14 min after RVD onset. Readmission of external Ca2+ always resulted in an increase
in [Ca2+]i, indicating that the lack of effect of external
Ca2+ readdition on the rate of RVD was not due to
block of Ca2+ entry during the regulatory response.
Similar results were obtained by adding the Ca2+ ionophore ionomycin (1 µM) during RVD (not shown).
These experiments do not disprove that an increase in
[Ca2+]i could have a role at earlier times during the
RVD response, as has been suggested for cell populations of renal proximal tubules (McCarty and O'Neil,
1992). Unfortunately, we cannot reintroduce Ca2+ at
earlier times and have a reliable measurement of the
change in RVD rates because there is not sufficient
sampling time to assess the change in the slope. The experiment is also not feasible using a two-hyposmotic-challenges protocol because the second response exhibits inactivation. Interestingly, we (Crowe et al., 1995
)
and others (Hoffmann et al., 1984
; Foskett et al., 1994
) have shown that elevating [Ca2+]i in cells maintained in
isosmotic medium produces cell shrinkage. On the basis of these results, we suggest that in addition to the
Ca2+-independent volume-regulatory machinery that is
activated by cell swelling in hyposmotic media, cells
also possess Ca2+-activated transport pathways that result in cell shrinkage in isosmotic media.
Ca2+ Signals in Osmotically Swollen Cells Loaded with Fura-2
The apparent increase in [Ca2+]i upon cell swelling
produced by hyposmotic solutions could be due to a
change in the K d of fura-2 for Ca2+ that accompanies a
decrease in internal ionic strength (Uto et al., 1991),
rather than a genuine increase in [Ca2+]i. Such a
change in internal ionic strength might be expected to
accompany a dilution of the internal constituents with
the hyposmotic challenge, but we have no independent
measure of such changes. We have two arguments to
suggest that the monitored Ca2+ signals are not spurious. First, we note that they disappear when the cells
are pretreated with BAPTA (Fig. 8). This argument is
somewhat weakened by the fact that the BAPTA might
essentially remove the free internal Ca2+ so that the
contention that the fura-2 affinity for Ca2+ becomes
higher is irrelevant since there would not be sufficient [Ca2+]i to react with the dye. However, the argument
becomes stronger for the case in which external Ca2+
was chelated with EGTA. In these cells, as in those
loaded with BAPTA, the apparent [Ca2+]i does not increase upon osmotic swelling, but actually decreases as
a consequence of intracellular dilution (Fig. 10). The
second argument is the demonstration that the Ca2+
signal is often absent in the second of two identical hyposmotic challenges even though the dilution of the
cell contents is the same as in the first hyposmotic challenge, which always results in a transient increase in intracellular Ca2+ (Fig. 6). The role, if any, of the rise of
[Ca2+]i observed during osmotic swelling in the presence of extracellular Ca2+ is not clear at this time. This
change in [Ca2+]i may be a modulating influence or
just an epiphenomenon in the RVD process.
A Signaling Role for Ca2+ in RVD?
Four pieces of evidence are usually given to propose an
active role for Ca2+ as the signal coupling cell volume
expansion to RVD responses (McCarty and O'Neil,
1992; Foskett, 1994
). Since these arguments are crucial
for the interpretation of the results of the present study, it is worth examining them. First, abolition of
RVD when a cell is swollen with a Ca2+-free hyposmotic
solution containing chelators is often given as proof of
a signaling role for Ca2+ (e.g., O'Connor and Kimelberg, 1993
). However, chelation in conjunction with
the osmotic shock is known to depolarize cells (Berman et al., 1994
). In addition, the osmotic challenge results in dilution of cell contents. All these factors, individually or in concert, could alter the driving forces for
ion efflux or shift the activation curves for voltage-sensitive channels involved in RVD as a consequence of
changes in surface potential. On the other hand, persistence of RVD upon external Ca2+ removal does not
exclude the possibility of an osmotically induced release of Ca2+ from intracellular stores (McCarty and
O'Neil, 1992
), which could be acting as the second
messenger.
The second argument generally offered to propose a
signaling role for Ca2+ on RVD is the disappearance of
the response upon external and internal Ca2+ chelation
with EGTA and BAPTA, respectively (e.g., Adorante
and Cala, 1995). The conclusions from this kind of experiment must also be treated with caution for the
same reasons discussed for external Ca2+ chelation.
Conversely, persistence of RVD in cells loaded with a
Ca2+ chelator has been interpreted as proof of a Ca2+-independent RVD (Lippmann et al., 1995
). However,
unless the adequacy of internal Ca2+ chelation is verified, as was done in the present study, this conclusion is
not warranted.
The third piece of evidence often given as proof for
Ca2+-dependent RVD is the block of the response with
Ca2+ entry blockers. In particular, these include blockers of voltage-gated Ca2+ channels (e.g., dihydropyridines) or blockers of mechanogated channels (e.g., gadolinium). However, such blockers may have effects on
mechanisms other than those involved in Ca2+ entry
(Foskett, 1994; Sánchez-Olea et al., 1995
; Hamill and McBride, 1996
).
The fourth argument usually given in favor of the Ca2+ hypothesis for RVD signaling is that hyposmotic swelling induces an increase in [Ca2+]i. Although a necessary logical condition for postulating a signaling role for Ca2+, it must be proved that the temporal relation between the change in [Ca2+]i and the onset of RVD are appropriate and, more importantly, that the Ca2+ increase is necessary for RVD and not just a parallel phenomenon.
Some investigators (e.g., Hoffmann et al., 1984; Cala,
1986) have suggested that swelling causes a change in
the Ca2+ affinity of a putative intracellular element of
the RVD machinery, and that therefore a rise in Ca2+ is
not necessary for Ca2+ to act as a trigger of the RVD response. First, if there was such a change in the Ca2+
sensitivity of some element of the RVD machinery, then
the second messenger or triggering signal would not be
Ca2+, but the change in affinity for Ca2+ of this putative
element of the RVD machinery. Second, we think that
this possibility is unlikely to explain our results because [Ca2+]i decreases below resting levels, yet hyposmotic
challenges elicit vigorous RVD responses.
Inactivation of RVD upon Sequential Hyposmotic Challenges
We show that the RVD response and the concomitant
increase in [Ca2+]i were abolished or significantly attenuated upon the second of two identical hyposmotic
challenges applied with an interval of 30-60 min. We
demonstrated that the RVD inactivation persists in the
nominal absence of external Ca2+ and is independent
of changes in internal Ca2+. Moreover, we show that inactivation is independent of the dye used to measure
cell volume changes. At least three explanations can be
offered for these observations. First, if the RVD is mediated by mechanogated channels, then it is conceivable
that these channels adapt and do not recover in time
for the second hyposmotic challenge (Hamill and
McBride, 1994). If there are mechanogated channels present in neuroblastoma cells that mediate Ca2+ influx in addition to those mediating volume regulatory
osmolyte effluxes (Falke and Misler, 1989
), then both
processes (i.e., the rise in intracellular Ca2+ and RVD)
would decrease in parallel without being interdependent. Second if RVD is mediated by the efflux of internal solutes from a finite store, then the apparent inactivation could represent the depletion of that store (Larson and Spring, 1984
). Third, inactivation could be
due to damage of RVD machinery as a consequence of
excessive mechanical stress (Hamill and McBride,
1997
). We have recently shown that RVD inactivation
can be reversed by loading the cells with taurine before
the second hyposmotic challenge. On the basis of this
observation, we suggest that inactivation is the consequence of depletion of some internal osmolyte store such as taurine (Brodwick et al., 1997
).
In summary, our simultaneous measurements of intracellular Ca2+ and volume in single neuroblastoma cells directly demonstrate that an increase in intracellular Ca2+ is not necessary for triggering RVD. In addition, we demonstrate that the RVD response "inactivates" upon repeated challenges, and that this process is also independent of Ca2+. Our results unify conflicting claims in the literature, based on indirect measurements in cell populations, by showing that: (a) RVD can be independent of a rise in intracellular Ca2+, and (b) there is a component of the response that is sensitive to Ca2+ chelation. The role of Ca2+ in the Ca2+- dependent component, if any, is not clear at this time. Ca2+ chelation may attenuate RVD through secondary effects. Nevertheless, it is clear that RVD itself can be triggered without a rise of [Ca2+]i in the neuronal cell lines investigated in this paper.
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FOOTNOTES |
---|
Address correspondence to Dr. F.J. Alvarez-Leefmans, Departamento de Neurobiología, Instituto Mexicano de Psiquiatría, Av. México-Xochimilco 101, México 22 D.F. C.P. 14370, México. Fax: 525-655-9980; E-mail: falvarea{at}mailer.main.conacyt.mx
Original version received 7 July 1997 and accepted version received 5 May 1998.
The authors thank Drs. Luis Reuss and Simon Lewis for their thoughtful comments on the manuscript, Mrs. Lynette Durant and Alicia Maldonado for secretarial work, Mr. José R. Fernández, Mr. Sergio Márquez Baltazar, and Miss Carrie Preite for skilled technical assistance, and Dr. William E. Crowe for participating in some experiments. Both cell lines used in this work were kindly provided by Dr. Marshall Nirenberg, Department of Biochemical Genetics, National Heart, Lung and Blood Institute (Bethesda, MD).
This work was supported by the National Institute of Neurological Disorders and Stroke grant NS29227, and National Council of Science and Technology (Mexico) grant F-285-N9209 to F.J. Alvarez-Leefmans.
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Abbreviations used in this paper |
---|
AM, acetoxymethyl ester; BAPTA, 1,2-bis-(o -aminophenoxy) ethane-N,N,N',N'-tetraacetic acid; CWV, cell water volume; RVD, regulatory volume decrease; SES, standard external solution.
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