EXPERIMENTAL CELL RESEARCH
ARTICLE NO.
227, 277–284 (1996)
0277
Hydrostatic Pressure to 400 atm Does Not Induce Changes in the
Cytosolic Concentration of Ca2/ in Mouse Fibroblasts:
Measurements Using Fura-2 Fluorescence
HUGH C. CRENSHAW1,2
AND
E. D. SALMON
Department of Biology, CB 3280, University of North Carolina, Chapel Hill, North Carolina 27599-3280
INTRODUCTION
Hydrostatic pressure has a pronounced effect on the
morphology and cytoskeletal organization of mammalian tissue cells. At pressures of about 300 atm (30
MPa), cells ‘‘round up’’—they withdraw their lamellar
extensions and greatly rearrange actin, tubulin, and
several other cytoskeletal proteins. It has been proposed that these changes are caused by a pressureinduced elevation of cytosolic Ca2/ concentrations. To
test this hypothesis we constructed a miniature optical
pressure chamber for fluorescent light microscopy to
allow measurement of cytosolic Ca2/ concentrations
with the intracellular fluorescent indicator fura-2.
This chamber and fura-2 were used to measure the
concentrations of Ca2/ in a mouse fibroblast line (C3H
10T1/2) at pressures up to 400 atm (40 MPa). Controls
included in vitro tests with standard buffers to determine the effect of pressure on fura-2 fluorescence.
These controls detected a change in fura-2 fluorescence with increasing pressure, but the data indicated
that pressure affects fura-2 fluorescence indirectly, by
altering the pH of the solution via pressure-induced
changes in the ionization of the pH buffer. These in
vitro changes in fura-2 fluorescence, nevertheless,
were small relative to changes in fura-2 fluorescence
produced by elevation in intracellular Ca2/ concentrations in response to physiological stimulation of the
cells (serum feeding after serum starvation). The
mouse fibroblasts rounded at pressures of 275 atm or
greater, as expected. However, no changes in cytosolic
Ca2/ concentrations were detected at any pressure, at
the onset of pressure, during periods of high pressure
(up to 10 min), or at the release of pressure. These
results strongly suggest that the mechanism by which
pressure alters cell morphology and cytoskeletal organization must, at least in these cells, be something
other than elevation of cytosolic Ca2/ concentrations.
q 1996 Academic Press, Inc.
1
Current address: Department of Zoology, Box 90325, Duke University, Durham, NC 27708-0325.
2
To whom correspondence and reprint requests should be sent.
Fax: (919) 684-6168; E-mail: [email protected].
Extreme hydrostatic pressures (bathymetric pressures) profoundly affect the physiology of surfacedwelling organisms [1]. These include such diverse effects as a positive inotropic effect on muscle contraction
at pressures greater than 10 atm (1 MPa) [2], high
pressure nervous syndrome which affects humans at
pressures exceeding 20 atm (2 MPa) [3], and disruption
of the cytoskeleton of vertebrate tissue cells at pressures above 250 atm (25 MPa) [4, 5].
The cytological and ultrastructural analyses of Marsland, Landau, Zimmerman, and Salmon (for reviews
see [4, 6, 7]) document that many cytoskeletal elements
are either depolymerized or reorganized by pressures
ranging from 200 to 400 atm. At pressures exceeding
about 300 atm, accompanying this disruption of cytoskeletal organization, many mammalian tissue cells
‘‘round up’’—they go from being flattened against their
substrate to being nearly spherical.
Several mechanisms can possibly explain pressure’s
alteration of cytoskeletal organization. One possible
mechanism is that pressure directly alters the dynamics of cytoskeletal assembly. Cytoskeletal elements are
polymeric proteins. Inside the cell these proteins are
in a steady-state equilibrium between monomer and
polymer. Pressure can affect the kinetics of polymer
assembly, driving equilibrium toward depolymerization when the molar volume of polymer is greater than
the molar volume of monomer [8, 9]. For example, increases in molar volume of approximately 100
mlrmol01 have been measured in vitro for the polymerization of myosin filaments [10–14], microtubules [15],
and actin microfilaments [16–19]. Pressure-induced
depolymerization of tubulin in the meiotic spindle of
an invertebrate egg closely follows the predictions of a
simple equilibrium model based on changes in the molar volume [15]. In other cells, however, pressure effects on the cytoskeleton are complex and are not directly predictable from in vitro analyses [4, 20].
A second possible mechanism is that pressure alters
some cellular control system that regulates the dynamics of cytoskeletal assembly. Otter et al. [4] proposed
277
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Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
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CRENSHAW AND SALMON
that pressure elevates cytosolic Ca2/ concentrations
and that this second messenger induces the observed
changes in cell morphology and cytoskeletal organization. Their proposal was based on the observations
that: (a) Many of pressure’s effects on the cytoskeleton,
especially microtubules, are consistent with elevated
cytosolic Ca2/ concentrations. (b) The behavior of Paramecium at elevated pressure resembles the behavior of
these ciliates when cytosolic Ca2/ is elevated, and a
membrane Ca2/ channel has been directly implicated
in pressure’s effects in these cells. (c) The mitotic spindle of invertebrates, which shortens when exposed to
high pressure, returns to its original length when held
for long durations at high pressure, indicating that
cells can adapt to pressure, which suggests that a regulatory mechanism, such as cytosolic Ca2/, is the pressure-sensitive mechanism.
The effects of pressure on cytosolic concentrations of
Ca2/, however, have not been reported. We have used
fura-2, a fluorescent indicator of cytosolic calcium concentrations, to measure cytosolic Ca2/ concentrations
in C3H 10T1/2 mouse fibroblasts at pressures up to 400
atm, pressures that induce cell rounding in this cell
type. Our measurements reveal no global, or wholecell, changes in the cytosolic concentrations of Ca2/ at
pressures from 1 to 400 atm at the onset of pressure,
while pressure is elevated, or when pressure is released. We also demonstrate that pressure does not
affect fura-2 fluorescence directly, but can alter fluorescence indirectly via pressure effects on the pH
buffer. These effects, however, are small. Thus Ca2/
regulation does not appear to be the target for pressure
disruption of cytoskeletal assembly for these cells.
MATERIALS AND METHODS
C3H 10T1/2 mouse fibroblasts were obtained from the Lineberger
Comprehensive Cancer Center (University of North Carolina–
Chapel Hill). The cells were cultured in DMEM (Sigma Chemical
Co.) supplemented with 10% calf serum (HyClone), 25 mM Hepes,
26 mM NaHCO3 (at equilibrium with 5% CO2), penicillin, and streptomycin (hereafter referred to as CMC). Culture conditions were pH
Ç7.2, 5% CO2 , 377C. Cells used in these experiments were cultured
on fragments (4 1 4 mm) of No. 1 coverslips that had been cleaned by
ultrasonic cleaning with sequential solutions of detergent (Alconox),
deionized water (four times), and 70% ethanol (two times).
A modified version of the microscope pressure chamber described
by Salmon and Ellis [24] was designed and constructed for these
experiments. Modifications included the use of sapphire windows
(thickness 1 mm; Edmunds Scientific) to permit UV excitation of the
fura-2 and changes in the dimensions of the vessel that decreased
the distance from the inside face of the window to the bottommost
face of the stainless steel support to 2.0 mm total.
A modified Nikon photoscan was used for the fluorescence measurements (Fig. 1). The photoscan is a photomultiplier-based measurement system mounted to a Nikon Diaphot inverted microscope
for measuring fluorescence from cells. Our modifications were: (a)
the fiber optic mount at the back of the microscope was modified
such that the face of the fiber optic was brought to focus at the back
aperture of the objective, (b) a diaphragm was added next to the
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FIG. 1. Diagram of the modified Nikon Photoscan used in these
experiments. A, Sapphire window of pressure chamber; B, Nikon
201/NA 0.75 Fluor objective; C, dichroic mirror with 380-nm cutoff;
D, bandpass filter centered on 515 nm with 50% transmission at 505
and 520 nm; E, heat-reflecting filter; F, photomultiplier; G, aperture
to block stray light mounted to inside of microscope filter cube housing; H, quartz heatblock filter; I, quartz lens; J, lump of clay used
to clamp fiber optic cable such that the face of the cable is both
centered on and in focus at the back aperture of the objective; K,
fiber optic; L, 340-nm bandpass filter; M, 380-nm bandpass filter; N,
optical chopper; O, 380-nm short-pass filter; P, 75-W xenon lamp.
(Asterisks denote components that are not normally part of the Photoscan system.)
filter cube to block stray light from the fiber optic from entering the
filter cube housing, and (c) heat block filters and bandpass filters
were placed in front of the photomultiplier to reduce background. A
Nikon 201, NA 0.75, Fluor objective was used for all measurements.
Although the working distance for this objective was too short to
focus on the cells, there was sufficient fluorescence from a field of
out-of-focus cells to make measurements. Excitation wavelengths
were 340 and 380 nm. Emission was measured at 500–525 nm. The
concentration of cytosolic Ca2/ was then estimated from the ratio of
fluorescence with excitation at 340 nm (fluorescence from the Ca2/bound fura-2) to fluorescence with excitation at 380 nm (fluorescence
from the Ca2/-free fura-2). The sample frequency in all experiments
was two ratio measurements per second.
The ratio was calculated as
R Å (F340 0 B340)/(F380 0 B380),
(1)
where R is the ratio, F340 is the fluorescence with 340-nm excitation,
B340 is the background fluorescence at 340-nm excitation, F380 is the
fluorescence with 380-nm excitation, and B380 is the background fluorescence at 380-nm excitation. Background fluorescence values were
measured using nonfluorescent buffers inside the chamber before
the addition of the sample.
Fura-2 calibration curves were generated using the hexapotassium
salt of fura-2 (Molecular Probes) at a concentration of 2.5 mM.
EGTA-buffered Ca2/ concentration standards were made with the
potentiometric technique described by Williams and Fay [25]. The
solutions were 10 mM EGTA, 100 mM KCl, 1 mM MgSO4 , 25 mM
NaCl, pH 7.2. (Note: The free Ca2/ concentrations determined by
this technique agreed within 10% with concentrations calculated by
the computer program Chelator [26].) Three different pH buffers
were used to identify the effect of pH buffer on fura-2 behavior at
pressure; these were (a) no pH buffer—allowing the EGTA to act as
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the pH buffer, (b) 10 mM Hepes, and (c) 8 mM sodium phosphate.
Fura-2 was tested at 1, 100, 200, 300, 400, and 500 atm.
The effects of pressure on fura-2 fluorescence in these Ca2/ buffers
was measured by filling the chamber with the test solution. The
optical path length of the chamber was about 1 mm. Under these
conditions, the fura-2 fluorescence was at least 3, and as much as 10,
times the background fluorescence at either excitation wavelength.
Cells were loaded using the acetoxymethyl ester of fura-2 (fura-2
AM) (Molecular Probes). We used a modification of the technique of
Roe et al. [27]. Cells were cultured to 40–50% confluency on the
coverslip fragments. One coverslip fragment was transferred to a
petri dish containing CMC with 4 mM NaHCO3 (at equilibrium with
air, hereafter referred to as CMA) at 47C for 10 min. Then the culture
medium was removed and replaced with the following loading medium: CMA, 50 mM fura-2 AM, 0.05% Pluronic F127. (The solvent
for the fura-2 AM and the Pluronic F127 was DMSO, yielding a final
concentration of 0.75% DMSO in the loading medium.) The cells were
incubated at 377C for 15 min. The cells were then rinsed two times
with phosphate-buffered saline (PBS) (6.5 mM Na2HPO4 , 1.5 mM
KH2PO4 , 2.7 mM KCl, 137 mM NaCl, 0.5 mM MgCl2 , 0.9 mM CaCl2)
containing 4.5 g/liter glucose and 0.1% bovine serum albumin (BSA).
The cells were then placed into the pressure chamber at 377C. The
medium inside the chamber also was PBS/glucose/BSA. All measurements commenced no sooner than 10 min after the final rinse, to
permit complete hydrolysis of the fura-2 AM to fura-2, and no later
than 15 min after the final rinse, to prevent intracellular compartmentalization of the fura-2. The cells were maintained at 377C at all
times after the loading procedure. Both visual inspection and the
selective permeabilization technique of Roe et al. [27] were used to
determine that almost all loading of the fura-2 was cytosolic and
that intracellular compartmentalization of the fura-2 was slight.
Background fluorescence was measured in the chamber before the
coverslip fragment carrying the cells was placed into the chamber.
For measurements with cells, background fluorescence was high,
relative to fluorescence from intracellular fura-2, due to the small
fluorescence from the fura-2 and the suboptimal configuration of the
optics. This background arose from a combination of reflections off
of the chamber windows, autofluorescence from the borosilicate glass
composing the upper window of the chamber (the window used for
transmitted illumination), and scattered light inside the microscope
and Photoscan assembly. Consequently, small changes in chamber
position had large effects (10–20%) on the measured background.
The fluorescence signal from fura-2 inside the cells was equal to, or
less than, the background and varied between experiments due both
to differences in the amount of fura-2 loading between experiments
and to the variable numbers of cells that appeared in any one field
of view. Background subtraction eliminated background from the
signal. However, background was measured before the addition of
cells, and the background changed before measurements on cells due
to small changes in the position of the chamber that occurred when
the cells were placed into the chamber. Due to this variation in the
background, combined with variation in the fluorescence signal from
the cells, the absolute value of the fluorescence ratio varied between
experiments, making comparisons of the fluorescence ratio between
experiments unreliable. Nevertheless, relative measurements within
a single experiment are reliable.
Two pressure regimens were used. In the first, the cells were observed at 1 atm for 30 s, then the pressure was elevated for 60 s,
after which the pressure was returned to 1 atm and the cells were
observed for another 30 s. Measurement of fluorescence was continuous throughout this 120-s period. The second regimen is similar to
the first; however, the cells were held at pressure for 300 s, and the
fluorescence was measured for 10 of every 60 s to reduce photobleaching of the fluor and photodamage to the cells. Cells were tested at
100, 200, 300, and 400 atm. Three groups of cells were tested at each
pressure.
Two positive controls were used to ensure that the system was
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capable of measuring changes in cytosolic Ca2/. In the first, cells
were serum starved for 24 h and then loaded with fura-2 as described
above (but using no serum at any step) and placed into the pressure
chamber with the top of the chamber removed. While measuring
fluorescence, calf serum was added to 10% final volume to ascertain
whether the system (the modified Nikon photoscan and the pressure
chamber) could measure the subsequent rise in cytosolic Ca2/ (see
[28]). In the second positive control, at the end of some of the pressure
experiments, the top of the chamber was removed and ionomycin
was added (5 mM final concentration) and the resultant rise in cytosolic Ca2/ was measured (see [27]). Note that removal of the upper
window (which contributed to the background as discussed earlier)
changed the background; however, the background measured with
the window in place was subtracted from the measurements made
after addition of ionomycin (in which the window was absent). This
has two consequences: (1) Comparisons between the ratio measured
before and measured after the addition of ionomycin should be
treated with care because the background subtraction alters the ratio. The effect here was to decrease the ratio measured after removal
of the window. This effect is conservative—the system was more
sensitive to changes in the ratio before removal of the window, i.e.,
any change in ratio before removal of the window would appear
larger than a change after removal of the window. (2) This caused
the ratio following ionomycin treatment to be smaller than that measured in the calf serum experiments.
Cells used for photography were fixed with formaldehyde at pressure as described by Bournes et al. [5], but without methanol extraction. The fixed cells were photographed using a Nikon FXA microscope with a 60X, NA 1.4, phase contrast objective and a MetaMorph
digital imaging system as described by Salmon et al. [29].
RESULTS
Effects of Pressure on Fura-2 in Vitro in Ca2/ Buffer
The fluorescence of fura-2 changed at the pressures
tested. However, the degree and direction of change
depended on the pH buffer used and on the concentration of free (unbound) Ca2/.
In the absence of a pH buffer, the effect of pressure
was much less than when either pH buffer, Hepes or
phosphate, was present (Fig. 2). With no pH buffer,
there was almost no change in the ratio for free Ca2/
concentrations of 30 and 300 nM (Fig. 2A). At 3 mM
free Ca2/, a concentration at which fura-2 is nearly
saturated, there was a larger effect, with the ratio decreasing linearly over the entire range of pressure.
With Hepes as the pH buffer, pressure caused a much
larger decrease in the ratio (Fig. 2B). This decrease was
detectable at all Ca2/ concentrations tested. Pressure
affected the fluorescence at both excitation wavelengths, except at the lowest Ca2/ concentrations tested
(10 and 22 nM) where no change was detected in fluorescence for 380-nm excitation. Again, the effect of
pressure was linear. Additionally, the decrease of the
ratio at higher pressures was larger for solutions with
higher free Ca2/ concentrations.
Conversely, with phosphate as the pH buffer, pressure caused the ratio to increase (Fig. 2C). Again, the
effect of pressure was linear, and it was larger when
the free Ca2/ concentration was higher—the ratio increased more at higher free Ca2/ concentrations.
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the ratio corresponds to a change in free Ca2/ of less
than 100 nM when pressure is constant (data not
shown). In our positive controls with serum-starved
cells at 1 atm, we observed changes in the fluorescence
ratio of 0.4–0.6 (Fig. 5A, see below). Thus pressureinduced changes in the ratio of fluorescence of fura-2,
at free Ca2/ concentrations less than 500 nM, were
always less than one-tenth of the changes expected
from rises of cytosolic Ca2/ in response to a physiological stimulus. This 10% error is acceptable for fura-2
measurements.
Effects of Pressure on Cells
FIG. 2. The effect of pressure on the fluorescence of fura-2 in
different pH buffers. Pressure changed the fluorescence of fura-2,
but the direction of change depended on the pH buffer used. All
solutions were 10 mM EGTA, 100 mM KCl, 1 mM MgSO4 , 25 mM
NaCl, pH 7.2. The numbers associated with each curve indicate the
concentration of free Ca2/ in nM at 1 atm pressure. The y axis is the
difference in the ratio measured at pressure (Ratio) subtracted from
the ratio measured at 1 atm (Ratio0). (A) No pH buffer. (B) The pH
buffer is 10 mM Hepes. (Note: For clarity, the curve for 22 nM free
Ca2/ is not shown because it overlaps the curve for 10 nM free Ca2/.)
(C) The pH buffer is 8 mM sodium phosphate.
We decided that this pressure effect on fura-2 was
not large enough to obscure our ability to observe
changes in cytosolic Ca2/. The reasoning is as follows:
The maximum change in the ratio observed in these
in vitro trials was approximately 00.2. This was with
Hepes as the pH buffer and with a free Ca2/ concentration of 10 mM. For free Ca2/ concentrations in physiological ranges for the cytosol (õ500 nM), the maximum
change was approximately 0.07 (positive for phosphate
buffer, negative for Hepes buffer). For free Ca2/ concentrations closer to that found in the cytosol of resting
cells (50–100 nM), the maximum observed change was
even smaller (approximately 0.04). A change of 0.07 in
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Cell morphology. C3H 10T1/2 fibroblasts rounded
at pressures above 275 atm (Fig. 3). This rounding occurs at similar pressures in, and is qualitatively similar
to, that observed for BSC-1 cells [5], HeLa cells [30],
rat osteosarcoma cells [30], fish keratinocytes, MG-63
human osteosarcoma cells, human foreskin fibroblasts,
and rat embryo fibroblasts (Crenshaw et al., unpublished data) that have been pressurized under similar
conditions.
Positive controls for measurement of cytosolic Ca2/.
It is known (e.g., [28]) that the addition of serum to
serum-starved tissue culture cells induces a transient
rise in cytosolic Ca2/ from resting concentrations below
100 nM to stimulated concentrations above 1000 nM.
Figure 4A presents the change in the fluorescence ratio
for our C3H 10T1/2 fibroblasts in response to serum
feeding after serum starvation. A large increase in the
measured fluorescence ratio is observed about 15 s
after the addition of serum. This expected change in
cytosolic Ca2/ concentration demonstrates that our
measurements are capable of detecting physiological
changes in cytosolic Ca2/.
Figure 4B presents the result of a pressure experiment in which the cells were held at 200 atm for 300
s, after which the cells were treated with ionomycin.
The large rise in the fluorescence ratio following addition of ionomycin indicates that the fura-2 inside these
cells was capable of detecting changes in cytosolic Ca2/
throughout these measurements.
Effects of Pressure on Cytosolic Ca2/
The results for all trials and both pressure regimens
are the same. There was no detectable change in the
ratio of fluorescence of fura-2 at any point in any of the
experiments. Figures 5A and 5B present the results of
experiments in which the cells were held at 400 atm
for 60 and for 300 s, respectively. The slow rise in the
ratio throughout the experiment in Fig. 5A is due entirely to photobleaching of the fura-2 and the resultant
differential effect of the background subtraction. The
trace in Fig. 5B is intermittent because the shutter
was only opened occasionally to reduce photobleaching.
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281
FIG. 3. Rounding of C3H 10T1/2 mouse fibroblasts at pressure. (A) Normal appearance of cells at 1 atm. (B) Appearance of cells after
15 m at 400 atm. After 2–3 min at 400 atm, cells begin to round—they retract the lamellipod and other cell processes. Most rounding
occurs in the first 5 min. At 300 atm, cells round more slowly, but appear qualitatively similar. (Scale bar, 50 mm.)
Again, there is no change in the fluorescence ratio at
the onset of pressure, during high pressure, at the release of pressure, or following the release of pressure.
pH buffer and, thus, the concentration of protons. The
resulting change in pH then alters the binding of EGTA
with Ca2/ because EGTA binds Ca2/ with the concomitant release of two protons [32],
DISCUSSION
EGTA / Ca2/ B 2H/ / Ca-EGTA,
These experiments provide strong evidence that
there is no change in cytosolic Ca2/ concentrations induced by hydrostatic pressures up to 400 atm, a pressure exceeding the pressure required to induce
rounding in C3H 10T1/2 fibroblasts. There was no
change in the ratio of fluorescence from fura-2 at any
time during any of the experiments at any of the tested
pressures.
These results are consistent with results obtained by
measuring cytosolic concentrations of Ca2/ in the eggs
of the sea urchin, Lytechinus pictus, with the fluorescent probe, fluo-3, at similar pressures (N. R. Gliksman
and E. D. Salmon, unpublished results). In a related
study, Philp [31] studied the effects of low pressure
(õ10 atm) narcotic gases (He, Xe, and N2O) on intracellular Ca2/ in platelets. He observed pressure-dependent changes in intracellular Ca2/ concentrations, but
the effects were gas dependent, so it is unclear how
these results relate to ours.
The effect of pressure on fura-2 fluorescence that we
observed in the in vitro buffers appears to be indirect.
One possible explanation is that pressure affects the
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(2)
making Ca2/ buffering by EGTA sensitive to pH
changes near 7 [33]. Any decrease in the pH of the
solution would drive this reaction to the left, raising
the concentration of free Ca2/ in the solution. The result would be an increase in the fluorescence ratio of
fura-2. Conversely, an increase in pH would cause the
ratio to decrease. This hypothesis is supported by four
lines of evidence: (1) Pressure has a much smaller effect
on the fluorescence ratio of fura-2 when only EGTA
with no pH buffer is used, (2) the effect of pressure
on the fluorescence ratio has opposite direction when
different pH buffers are added, (3) the directions of
change in pH predicted by the changes in the fluorescence ratio are in agreement with published changes
in pH for phosphate- and Hepes-buffered solutions (see
below), and (4) the effect of pressure on the fluorescence
ratio was higher for higher concentrations of free Ca2/
for all the buffers tested.
Expanding on the third line of evidence, Eq. (2) suggests that the rise in the fluorescence ratio with phosphate buffer is due to a decrease in pH when the phos-
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phate buffer is at pressure. Phosphate buffers do decrease pH and, in fact, have an unusually large change
in molar volume (22 mlrmol01 [34]) such that a solution
buffered with phosphate near neutrality at 1 atm decreases about 0.3 pH at 500 atm [34, 35]. The decrease
in the fluorescence ratio with Hepes buffer at pressure
indicates that Hepes-buffered solutions increase pH at
pressure. We could only find one report of pressureinduced pH changes in Hepes-buffered solutions at
pressure. Jaenicke [35] reports an increase of approximately pH 0.02/500 atm for a complex culture medium
with 120 mM Hepes. This change in pH is in the same
direction as, but of much smaller magnitude than, the
changes suggested by the changes we observed in the
fluorescence ratio of fura-2. Our results suggest that
the change in pH with Hepes, as a function of pressure,
in the simple ionic solution used in these experiments,
has opposite direction, but similar magnitude, to the
change in pH occurring with phosphate. (Again, the
FIG. 5. The fluorescence ratios from fura-2 in cells exposed to
400 atm (40 MPa) pressure. (A) C3H 10T1/2 cells were held at 400
atm for 60 s and then the pressure was returned to 1 atm. The
pressure rose to 400 atm at 30 s and returned to 1 atm at 90 s. No
change in the ratio is detectable. (B) Cells were held at 400 atm for
5 min and then the pressure was returned to 1 atm. The pressure
rose at 30 s and returned to 1 atm at 330 s. The ratio was measured
only for 10 s at the beginning of each 1-min period to reduce photobleaching of the fura-2 and photodamage to the cells. Again, no
change in the ratio is detectable.
FIG. 4. Positive controls for in vivo Ca2/ measurements. (A) The
change in the fluorescence ratio in serum-starved cells following addition of calf serum to the medium. 10% calf serum (final concentration, volume/volume) and 5 mM ionomycin (final concentration) were
added at the indicated time points. The large rise in the ratio following addition of calf serum is indicative of elevated cytosolic Ca2/
concentrations. The larger rise in the ratio following the addition of
ionomycin indicates that the fura-2 inside the cell was competent to
measure larger increases in cytosolic Ca2/ concentrations. (B) The
change in the fluorescence ratio of cells due to the addition of 5
mM ionomycin (final concentration) after a pressure experiment (200
atm). The rise in the ratio indicates that the cells were regulating
Ca2/ at low levels throughout the pressure experiment and that the
fura-2 was competent to measure increases in cytosolic Ca2/ concentrations at the end of the experiment. (Note that the difference in
the baseline ratios and in the ratios measured after the addition of
ionomycin in these two controls arise from differences in background
subtraction in the two experiments, as discussed under Materials
and Methods.)
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culture medium used by Jaenicke had many other molecules, including phosphate, that would act as buffers,
so their measured changes in pH with Hepes cannot
be directly compared to our results.)
The fourth line of evidence suggests that pressure’s
effect on the fluorescence ratio arises from driving Ca2/
off of EGTA for a decrease in pH [driving Eq. (2) to the
left] or driving Ca2/ onto EGTA for an increase in pH.
This effect will be more pronounced in a solution with
a higher concentration of Ca2/ because Ca2/ cannot
be forced on or off EGTA if there is no Ca2/ present.
Accordingly, we do see larger pressure effects in solutions (for all buffers) with higher Ca2/ concentrations.
One final point is that fura-2 is an EGTA-like molecule [36]. Thus pH also will alter Ca2/ binding to fura2 in a manner similar to Eq. (2), so the pH changes at
pressure might be expected also to change the fluorescence ratio directly (not just by altering the buffering
of Ca2/ by EGTA). However, fura-2 is more closely related to BAPTA [36], which is much less sensitive to
pH variations near 7 [33]. Our measurements were
made at pH 7.2, so variations in pH caused by pressure’s effect on the pH buffers would strongly affect
EGTA’s binding with Ca2/ but should have a smaller
effect on fura-2’s binding with Ca2/. Thus the changes
in the fluorescence ratio of fura-2 in the presence of pH
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FURA-2 MEASUREMENTS OF Ca2/ UNDER HYDROSTATIC PRESSURE
buffers are probably due to pressure’s effect on the pH
buffer.
Regardless, the effects of pressure on the fluorescence ratio of fura-2 should not have interfered with
our ability to measure physiological ranges of cytosolic
Ca2/. As stated earlier, the largest changes we observed in vitro for the ratio, with either phosphate or
Hepes as the pH buffer, was a change that corresponded to a change in cytosolic Ca2/ of less than 100
nM. Changes of cytosolic Ca2/ in serum-starved cells,
responding to serum, yielded changes in the ratio indicative of an elevation of cytosolic Ca2/ on the order of
1000 nM, so the effect of pressure on the ratio should
have been small relative to changes caused by physiological changes in cytosolic Ca2/.
It is curious, nevertheless, that we observed no
change in the fluorescence ratio of fura-2 in our experiments. We expected at least to see some direct effect
of pressure on the fluorescence of fura-2; however, if
such an effect exists inside the cell, it is small.
Another explanation for the absence of any pressure
effect on fura-2 in our measurements of cells under
pressure is that changes in the fluorescence ratio of
fura-2 caused by pressure were exactly offset by
changes in the cytosolic concentration of Ca2/. This is
unlikely; however, if this did occur, the changes in the
cytosolic concentration of Ca2/ were much smaller than
the 1000 nM changes that occur in response to serum
following serum starvation, as discussed above.
Our results demonstrate that hydrostatic pressures
up to 400 atm do not cause large, global changes in the
cytosolic concentrations of Ca2/. It remains possible
that small changes (õ100 nM) occur or that Ca2/ concentration gradients inside the cell are disrupted because our techniques were unable to resolve such
changes. Nevertheless, no changes similar to those
changes that arise in response to physiological stimuli,
e.g., exposure to serum following serum starvation or
exposure to mitogens [28, 37, 38], were observed.
Much of the argument of Otter et al. [4] remains,
although the mechanism they propose, pressure elevation of cytosolic Ca2/, does not appear correct for mammalian tissue cells. Most notably, the point first put
forward by Salmon [15], that some cytoskeletal changes
appear to adapt to pressure, remains enigmatic and
strongly suggests that some intracellular regulator of
cytoskeletal organization is affected by pressure. Crenshaw et al. [30] describe differential sensitivity of cytokeratin intermediate filaments and provide additional
evidence that suggests pressure might be acting upon
a regulatory pathway. Nevertheless, direct evidence
identifying any regulatory mechanism of cell physiology or cytoskeletal organization is not yet available.
We thank Shannon Davis and William Davis (University of North
Carolina–Chapel Hill) for assistance in the early stages of this proj-
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ect. Helpful comments were provided by Albert Harris, Steven Parsons, Vicki Skeen, Robert Skibbens, Phong Tran, and Jennifer Waters. This project was supported by the U.S. Office of Naval Research,
Grant N00014-92-J-1504.
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Received December 26, 1995
Revised version received May 29, 1996
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