Cell volume control in Paramecium: factors that activate the control mechanisms
Pacific Biomedical Research Center, Snyder Hall 306, University of Hawaii at Manoa, 2538 The Mall, Honolulu, HI 96822, USA
Author for correspondence (e-mail:
naitoh{at}pbrc.hawaii.edu)
Accepted 30 November 2004
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Summary |
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Key words: cell volume control, osmoreception, osmoregulation, RVI, RVD, cytosolic pressure, osmotic pressure, bulk modulus, contractile vacuole, Paramecium
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Introduction |
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Conversely, the contractile vacuole complex (CVC) has been regarded to be
the organelle responsible for osmoregulation in fresh water protozoans and
fresh water sponges (Jepps,
1947; Kitching,
1967
; Dunham and Kropp,
1973
; Patterson,
1980
; Allen and Naitoh,
2002
). As a matter of fact, the amount of fluid discharged from
the contractile vacuole (CV) to the cell's exterior markedly increases when
the cell is exposed to a hypotonic solution, while the fluid discharge ceases
when the cell is exposed to an isotonic or hypertonic solution. Dunham and
Kropp (1973
) suggested the
presence of cell volume control mechanisms in Tetrahymena, a fresh
water ciliate, in addition to the CVC. It is, therefore, interesting and
important to know how much the CVC contributes to the osmoregulation or volume
control in the Paramecium cell.
In the present study, we examined the effects of a change in the external
osmolarity on both cell volume and the amount of fluid discharged from the CV
in P. multimicronucleatum. We give evidence for the presence of two
hypothetical osmolyte transport mechanisms in Paramecium that are
responsible for regulatory volume increase (RVI) and regulatory volume
decrease (RVD), respectively (Strange,
1994; Hoffmann and Dunham,
1995
; Lang et al.,
1998
; Baumgarten and Feher,
2001
). These two transport mechanisms fill the major role in
regulating cell volume and the cytosolic osmolarity. The CVC seems to fill a
lesser role in cell volume regulation.
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Materials and methods |
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Experimental solutions
All the experimental solutions, each with a different osmolarity
(4204 mosmol l-1), were made by adding different amounts of
sorbitol to a standard saline solution that contained (mmol l-1 in
final concentration) 2.0 KCl, 0.25 CaCl2 and 1.0-MOPS-KOH (pH 7.0).
Osmolarity of the standard saline solution without sorbitol was 4 mosmol
l-1 (Naitoh et al.,
1997a). In some experiments, a 30 mmol l-1 KCl or 10
mmol l-1 tetraethylammonium (TEA) chloride-containing solution was
used. The osmolarity of each solution was determined by using a freezing-point
osmometer (Advanced Instruments, Inc., Norwood, MA, USA).
Experimental procedures
An experimental trough (18 mm in length) with a rectangular cross section
(0.2 mm in depth, 6 mm in width) was made from cover glass. The trough was
first filled with 0.02% (v/v) poly-L-lysine (Sigma, St Louis, MO, USA)
solution, then cells suspended in an adaptation solution were introduced into
the trough from its one end, while excess solution in the trough was removed
from the other end by using filter paper. Cells that adhered to the bottom
surface of the trough were subjected to a change in the external osmolarity.
This was done by introducing an experimental solution with an osmolarity
different from the adaptation solution into the trough as described above.
Images of the cells were magnified through a phase contrast objective
(x40) of an inverted microscope (Olympus America Inc., Lake Success, NY)
and video-recorded by using a CCD camera (Dage MIT Inc., Michigan City, IN)
and a video cassette recorder (Sony Corp., Park Ridge, NY) at 30 frames
s-1.
Estimation of cell volume and its relative change
Video-recorded images of a cell were fed into a computer (Apple Computer
Inc. Cupertino, CA) and the area (A) and length (l) for each
image were determined by using NIH Image (downloaded from
http://rsb.info.nih.gov/nih-image/).
If we assume that a Paramecium cell approximates a solid of
revolution, the volume of the cell (v) can be approximated by an
equation as,
![]() | (1) |
![]() | (2) |
![]() | (3) |
![]() | (4) |
![]() | (5) |
![]() | (6) |
Determining the rate of fluid discharge from the CV
The rate of fluid discharge from the CV
() was determined
on the replayed images of the CV.
was obtained by
dividing the maximum volume of the spherical CV immediately before the start
of discharge, that is calculated from the CV diameter, by the period of time
from the start of fluid filling immediately after the previous fluid discharge
to the start of the present fluid discharge, i.e. the time between two
successive fluid discharges (Naitoh et
al., 1997a
; Stock et al.,
2001
).
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Results |
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Time course of change in cell volume after changing the external osmolarity
Decreasing the osmolarity
Five groups of Paramecium cells adapted to different osmolarities,
i.e. 164, 144, 124, 84 or 64 mosmol l-1, for 18 h were subjected to
a 60 mosmol l-1 decrease in the external osmolarity and the time
course of change in cell volume for each group was determined, and shown in
Fig. 2. Each cell volume is
presented as a value relative to the cell volume before decreasing the
external osmolarity (at time 0).
|
The volume of 164 mosmol l-1-adapted cells increased to its peak
value of 1.07 times the initial volume (at time 0) in
10 min after
decreasing the external osmolarity to 104 mosmol l-1
(Fig. 2A). The volume then
decreased to its initial value in
30 min after decreasing the external
osmolarity and continued to decrease to the lowest value of
0.95 by
50 min. The volume then tended to increase slowly.
The volume of 144 mosmol l-1-adapted cells increased to a
plateau value of 1.20 times the initial volume in
20 min after
decreasing the external osmolarity to 84 mosmol l-1
(Fig. 2B). In contrast to the
previous case of 164 mosmol l-1-adapted cells, the cells remained
swollen at this plateau level after that. A series of three pictures of a
representative 144 mosmol l-1-adapted cell taken at 0, 15 or 30 min
after decreasing the external osmolarity is shown in
Fig. 4A. The cell at 15 min was
thicker and longer than the cell at 0 min, and the cell at 30 min was thicker
and longer than that at 15 min.
|
The volume of 124 mosmol l-1-adapted cells increased to its peak
value of 1.23 times the initial volume in 30 min after decreasing the
external osmolarity to 64 mosmol l-1. The volume then decreased to
resume its initial value (Fig.
2C).
As is shown in Fig. 2D, the
volume of 84 mosmol l-1-adapted cells increased to its peak value
of 1.14 in
10 min after decreasing the external osmolarity to 24
mosmol l-1. The volume then decreased to its initial value at time
0 in
30 min and continued to decrease to
0.95 in 40 min. The volume
then tended to increase slowly. A series of three pictures of a representative
84 mosmol l-1-adapted cell taken at 0, 15 or 30 min after
decreasing the external osmolarity, is shown in
Fig. 4B. The cell at 15 min was
thicker and longer than the cell at 0 min, while the cell at 30 min was
thinner than the cell at 15 min.
As is shown in Fig. 2E, the
volume of 64 mosmol l-1-adapted cells increased to a plateau value
of 1.17 in 20 min after decreasing the external osmolarity to 4 mosmol
l-1. The cells remained swollen at this level after that.
Increasing the osmolarity
Five groups of Paramecium cells adapted to different osmolarities,
i.e. 104, 84, 64, 24 or 4 mosmol l-1, for 18 h, were subjected to a
60 mosmol l-1 increase in the external osmolarity and the time
course of change in cell volume for each group was determined and shown in
Fig. 3.
|
The volume of 104 mosmol l-1-adapted cells decreased to its
lowest value of 0.84 times the initial volume in
10 min after
increasing the external osmolarity to 164 mosmol l-1
(Fig. 3A). The volume then
increased to
0.95 in 60 min after increasing the external osmolarity.
The volume of 84 mosmol l-1-adapted cells decreased to its
lowest value of 0.74 times the initial volume in
20 min after
increasing the external osmolarity (Fig.
3B). In contrast to the previous case of 104 mosmol
l-1-adapted cells, the cells remained shrunken at this lowest level
after that.
The volume of 64 mosmol l-1-adapted cells decreased to its
lowest value of 0.65 times the initial volume in
10 min after
increasing the external osmolarity to 124 mosmol l-1
(Fig. 3C). The volume then
increased to
0.85 in
60 min after increasing the external
osmolarity. A series of three pictures of a representative 64 mosmol
l-1-adapted cell taken at 0, 15 or 30 min after increasing the
external osmolarity to 124 mosmol l-1 is shown in
Fig. 4D. A marked indentation
was seen in the posterior one third of the cell at 15 min. Thisindentation was
less pronounced in the cell at 30 min.
The volume of 24 mosmol l-1-adapted cells decreased to its
lowest value of 0.80 times the initial volume in
10 min after
increasing the external osmolarity to 84 mosmol l-1
(Fig. 3D). The volume then
increased to
0.99 in 40 min after increasing the osmolarity and tended to
decrease slowly after that.
The volume of 4 mosmol l-1-adapted cells decreased to its lowest
value of 0.75 times the initial volume in
40 min after increasing
the external osmolarity to 64 mosmol l-1 and remained unchanged
after that (Fig. 3E). A series
of three pictures of a representative 4 mosmol l-1-adapted cell
taken at 0, 15 or 30 min after increasing the external osmolarity to 64 mosmol
l-1 is shown in Fig.
4C. The cell at 15 min was thinner and shorter than the cell at 0
min, and the cell at 30 min was shorter than the cell at 15 min.
Effects of the degree of change in the external osmolarity on the time course of change in cell volume
To know the effects of the degree of change in the external osmolarity on
the time course of change in cell volume after changing the external
osmolarity, the time courses were compared among different groups of cells
adapted to the same osmolarity and then subjected to different changes in
osmolarity.
Decreasing the osmolarity
When 144 mosmol l-1-adapted cells were subjected to 104 mosmol
l-1, where osmolarity decrease was 40 mosmol l-1, cell
volume increased to a plateau value of 1.05 in
10 min and remained
unchanged at this level after that (Fig.
5A, black open circles). This time course was essentially the same
as that seen after their subjection to 84 mosmol l-1, where
osmolarity decrease was 60 mosmol l-1
(Fig. 2B; the same data is
shown in Fig. 5A by gray open
circles for the sake of comparison), but the plateau value was far smaller
than that for the 60 mosmol l-1 decrease (
1.20).
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When 84 mosmol l-1-adapted cells were subjected to 44 mosmol
l-1, where the osmolarity decrease was 40 mosmol l-1
(black open circles) or to 64 mosmol l-1, where osmolarity decrease
was 20 mosmol l-1 (red open circles), cell volume increased to a
peak value of 1.14, then decreased to resume its initial value
(Fig. 5B). These time courses
are essentially the same with that seen after their subjection to 24 mosmol
l-1, where osmolarity decrease was 60 mosmol l-1
(Fig. 2D; also shown in
Fig. 5B by gray open circles
for the sake of comparison). The peak values were almost the same among these
three cases (
1.14) independently of the total decrease in the external
osmolarity. However, the time to reach the peak was shorter when the decrease
in the external osmolarity was larger, i.e. the time was
10, 20 and 30
min for a 60, 40 and 20 mosmol l-1 decrease, respectively.
Increasing the osmolarity
When 4 mosmol l-1-adapted cells were subjected to 44 mosmol
l-1, where osmolarity increase was 40 mosmol l-1, cell
volume decreased to its lowest value of 0.79 in
40 min and then
remained almost unchanged at this level
(Fig. 6A, black open circles).
This time course is essentially the same with that seen after their subjection
to 64 mosmol l-1, where osmolarity increase was 60 mosmol
l-1 (Fig. 3E; also
shown in Fig. 6A by gray open
circles for the sake of comparison). However, decrease in the volume was
smaller than that for a 60 mosmol l-1 decrease (
0.75).
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When 64 mosmol l-1-adapted cells were subjected to 84 mosmol
l-1 (Fig. 6B), where
osmolarity increase was 20 mosmol l-1 (black open circles), or to
104 mosmol l-1, where osmolarity increase was 40 mosmol
l-1 (red open circles), cell volume first decreased to its lowest
value, then increased to resume its initial value. These time courses are
essentially the same with that seen after their subjection to 124 mosmol
l-1, where osmolarity increase was 60 mosmol l-1
(Fig. 3C; also shown in
Fig. 6B by gray open circles
for the sake of comparison). However, the degree of volume decrease was larger
when osmolarity increase was larger, i.e. the lowest volume was 0.91,
0.80 and 0.64 for an osmolarity increase of 20, 40 and 60 mosmol
l-1, respectively.
Effects of TEA+ and K+ in the external solution on cell volume change
The major osmolytes in the cytosol of Paramecium are K+
and Cl- (Akita,
1941; Stock et al.,
2002
). It is, therefore, highly probable that a K+
transport system(s) in the plasma membrane is involved in restoring cell
volume in Paramecium (see Fig.
2A,C,D; Fig.
3A,C,D; Fig. 5B;
Fig. 6B). We, therefore,
examined the effect of the presence of 10 mmol l-1 TEA+,
a potent K+ channel inhibitor, or excess (30 mmol l-1
instead of a normal 2 mmol l-1) K+ in the external
solution on the time course of the change in cell volume after changing the
external osmolarity (Fig.
7).
|
Decreasing the osmolarity
164 mosmol l-1-adapted cells continued to swell after decreasing
the external osmolarity to 104 mosmol l-1 in the presence of 10
mmol l-1 TEA+ (Fig.
7A; black open circles) or 30 mmol l-1 K+
(Fig. 7B; black open circles)
in contrast to the control cells that returned to their initial volume after a
temporary swelling (Fig. 7A,B;
gray circle data from Fig.
2A).
Increasing the osmolarity
In the presence of 10 mmol l-1 TEA+, 104 mosmol
l-1-adapted cells shrank to a more-or-less stationary volume of
0.75 after increasing the external osmolarity to 164 mosmol
l-1 and did not return to their initial volume
(Fig. 7C; black open circles);
the cells returned to their initial volume after a temporary shrinking in the
absence of TEA+ (Fig.
7C; gray circles data from Fig.
3A).
Conversely, the cells returned to their initial volume after they showed a
temporary shrinking upon increasing the external osmolarity to 164 mosmol
l-1. They responded more quickly in the presence of 30 mmol
l-1 K+ (Fig.
7D; black open circles) than in the presence of a normal 2.0 mmol
l-1 K+ (Fig.
7D; gray circles data from Fig.
3A). The time needed to return to the initial volume was 20
min as opposed to 60 min or more in the control. The degree of shrinking was
smaller (
0.90) than for the control (
0.84).
No change in the osmolarity
To determine whether the presence of TEA+ or excess
K+ in the external solution itself affects cell volume, the time
course of change in volume of the cells adapted to 164 mosmol l-1
after they were transferred into a 10 mmol l-1 TEA+ or a
30 mmol l-1 K+-containing 164 mosmol l-1
solution was determined. The cells were not subjected to a change in the
external osmolarity. Cell volume increased very slowly to 1.05 in 60 min
after they were subjected to 10 mmol l-1 TEA+. Cell
volume scarcely changed after the cells were subjected to 30 mmol
l-1 K+-containing solution (data not shown).
Change in the rate of fluid discharge from the CV, CVC, after changing the external osmolarity
To examine how much the CV affects cell volume, the time course of change
in the rate of fluid discharge,
(Naitoh et al., 1997a
), after
changing the external osmolarity was determined. Four representative results
are shown in Fig. 8,where
is presented as
a value relative to its initial value before changing the external
osmolarity.
|
Decreasing the osmolarity
When a 164 mosmol l-1-adapted cell was transferred into a 104
mosmol l-1 solution,
increased to its
peak value of
6.5 in
13 min after the transfer, then decreased to a
more-or-less stationary value of
2.5
(Fig. 8A). The initial value
for
in this cell
was
11.9 fl s-1.
When a 144 mosmol l-1-adapted cell was transferred into a 84
mosmol l-1 solution,
increased to a
plateau value of
6.5 in
13 min after the transfer.
then decreased
gradually with time (Fig. 8B).
The initial value for
in this cell was
9.2 fl s-1.
Increasing the osmolarity
When a 104 mosmol l-1-adapted cell was transferred into a 164
mosmol l-1 solution, the cell suddenly ceased its CVC activity so
that became 0 in
less than 10 min after the transfer.
activity started
to recover gradually
30 min after the transfer
(Fig. 8C). The initial value
for
in this cell
was
22.7 fl s-1. The similar time course was observed when a
84 mosmol l-1-adapted cell was transferred into a 144 mosmol
l-1 solution (Fig.
8D). The initial value for
in this cell was
12.7 fl s-1.
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Discussion |
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An osmotic pressure difference between the cytosol and the adaptation solution balances a hydraulic pressure within the cytosol in a cell adapted to a particular osmolarity
We previously found that the cytosolic osmolarity of a Paramecium
cell following adaptation to an osmolarity in a range from 4 to 204 mosmol
l-1 always became higher than the osmolarity of the adaptation
solution (Stock et al., 2001).
This implies that an adapted Paramecium cell is always swollen to an
extent determined by the osmotic pressure difference between the cytosol and
the adaptation solution (
cyt). This osmotic pressure difference
balances a hydraulic pressure in the cytosol, the `cytosolic pressure'
(Pcyt), which is generated by an elastic membrane
surrounding the cell as the cell becomes osmotically swollen. If either one or
both of these pressures are somehow modified, the cell volume changes until a
new balance between these pressures is established.
The presence of a cytosolic pressure in adapted cells was clearly
demonstrated when 4 mosmol l-1-adapted cells were subjected to 64
mosmol l-1 (Fig. 3E)
or 44 mosmol l-1 (Fig.
6A). In these cases, the cells shrank even though the cytosolic
osmolarity of 4 mosmol l-1-adapted cells, i.e. 75 mosmol
l-1 (Stock et al.,
2001
), was higher than the osmolarity to which the cells were
subjected, i.e. 44 or 64 mosmol l-1. The cytosolic pressure of 4
mosmol l-1-adapted cells exceeded the osmotic pressure difference
that was lowered by raising the external osmolarity, and thereby compressed
the cells. Similarly, 84 mosmol l-1-adapted cells shrank when they
were subjected to 144 mosmol l-1
(Fig. 3B), even though the
cytosolic osmolarity of 84 mosmol l-1-adapted cells, i.e.
160
mosmol l-1 (Stock et al.,
2001
), was higher than 144 mosmol l-1. We previously
demonstrated the presence of a cytosolic pressure in Paramecium cells
was the basis for the discharge of the contractile vacuole fluid (Naitoh et
al.,
1997a
,b
).
Cell volume control mechanisms are activated when the external osmolarity is changed beyond a critical osmolarity
We previously found that the cytosolic osmolarity of the
Paramecium cell changed stepwise at around 75 or 160 mosmol
l-1 when the adaptation osmolarity was changed gradually
(Stock et al., 2001). This
implies that an active change in the cytosolic osmolarity takes place when the
external osmolarity is changed beyond these osmolarities. Hereafter, these two
osmolarities will each be termed a `critical osmolarity'
(CN) for causing an active change in the cytosolic
osmolarity.
It can, therefore, be predicted that when the external osmolarity is decreased beyond a critical osmolarity, an osmolyte-transport mechanism(s) in the plasma membrane is activated to decrease the cytosolic osmolarity, so that a cell that has been swollen by its subjection to a decreased osmolarity may resume its initial volume. When the external osmolarity is increased beyond a critical osmolarity, another osmolyte-transport mechanism(s) is activated to increase the cytosolic osmolarity, so that the cell that has been shrunken by its subjection to an increased osmolarity may resume its initial volume. By contrast, when the external osmolarity is changed within an osmolarity range where no critical osmolarity is included, the cell will remain swollen or shrunken, since the osmolyte transport mechanisms are not activated.
These predictions proved correct. That is, cell volume recovery from its temporary swelling caused by a decrease in the external osmolarity was observed when the decrease took place beyond a critical osmolarity, i.e. a decrease from (in mosmol l-1) 164 to 104 (Fig. 2A; the critical osmolarity is 160), that from 124 to 64 (Fig. 2C; the critical osmolarity is 75), that from 84 to 24 (Fig. 2D; the critical osmolarity is 75), that from 84 to 44 (Fig. 5B; the critical osmolarity is 75) and that from 84 to 64 (Fig. 5B; the critical osmolarity is 75). Leftward arrows with a red asterisk in Fig. 9B correspond to these experiments. Each arrow crosses with either one or the other of two red vertical lines that corresponds to a critical osmolarity of 75 or 160 mosmol l-1, respectively. By contrast, cells remained swollen when a decrease in the external osmolarity took place within an osmolarity range where no critical osmolarity was included, i.e. a decrease from (in mosmol l-1) 144 to 84 (Fig. 2B), that from 144 to 104 (Fig. 5A) and that from 64 to 4 (Fig. 2E). Leftward arrows without an asterisk in Fig. 9B correspond to these experiments. These arrows cross neither one or the other of the two red vertical lines.
Paramecium showed recovery from its temporary shrinkage after its subjection to an increase in the external osmolarity when the increase took place beyond a critical osmolarity, i.e. an increase from (in mosmol l-1) 104 to 164 (Fig. 3A; the critical osmolarity is 160), that from 64 to 124 (Fig. 3C; the critical osmolarity is 75), that from 64 to 104 (Fig. 6B;the critical osmolarity is 75), that from 64 to 84 (Fig. 6B; the critical osmolarity is 75) and that from 24 to 84 (Fig. 3D; the critical osmolarity is 75). Rightward arrows with a blue asterisk in Fig. 9B correspond to these experiments. These arrows cross either one or the other of the two red vertical lines. By contrast, the cell remained shrunken when an increase in the external osmolarity took place within an osmolarity range where no critical osmolarity was included, i.e. an increase from (in mosmol l-1) 84 to 144 (Fig. 3B) and that from 4 to 64 (Fig. 3E). Rightward arrows without an asterisk in Fig. 9B correspond to these experiments. These arrows cross neither of the two red vertical lines.
Thus two hypothetical osmolyte transport mechanisms are proposed for
Paramecium, i.e. (1) responsible for cell volume recovery from
osmotic swelling and (2) responsible for cell volume recovery from osmotic
shrinkage. These hypothetical transport mechanisms may correspond to osmolyte
transport mechanisms found in the cells of multicellular animals that are
responsible for (1) regulatory volume decrease, RVD, and for (2) regulatory
volume increase, RVI, respectively
(Strange, 1994;
Hoffmann and Dunham, 1995
;
Lang et al., 1998
;
Baumgarten and Feher,
2001
).
Bulk modulus of the cell changes as the cytosolic osmolarity changes
It was predicted that a cell adapted to an osmolarity very close to a
critical osmolarity for activation of RVI, i.e. 75 mosmol l-1 in an
osmolarity range of less than 75 mosmol l-1 or 160 mosmol
l-1 in an osmolarity range from 75 to 160 mosmol l-1,
shows neither osmotic swelling nor shrinkage, since the cytosolic osmolarity
is nearly the same as the critical osmolarity (see
Fig. 9A). Hereafter, the volume
of a cell under this condition will be termed the `natural cell volume'
(vN). The natural cell volume is assumed to be determined
by the structural and mechanical properties of the cytoskeleton and the
associated pellicular membranes.
Conversely, the volume of a cell adapted to an osmolarity lower than the critical osmolarity is larger than the natural cell volume, since the external osmolarity is lower than the cytosolic osmolarity, so that the cell will be osmotically swollen. The cell volume becomes maximum when the adaptation osmolarity is the lowest in an osmolarity range where no critical osmolarity is included, since the difference in the osmolarity between the cytosol and the external solution becomes maximum. It was, therefore, predicted that the cell volume might change stepwise at each critical osmolarity as the adaptation osmolarity changes.
This prediction, however, proved incorrect. As is shown in Fig. 1, the cell volume of 4 mosmol l-1-adapted cells was significantly (P<0.01) larger than those cells adapted to an osmolarity higher than 4 mosmol l-1, i.e. (in mosmol l-1) 64, 84, 144, 164 or 204, whereas there is no significant difference in cell volume between 64 mosmol l-1- and 84 mosmol l-1-adapted cells (P=0.691, N=5664), or between 144 mosmol l-1- and 164 mosmol l-1-adapted cells (P=0.158, N=9596). These facts imply that cell volume changes continuously without showing a stepwise change at each critical osmolarity as the adaptation osmolarity changes.
Cell volume (v) plotted against adaptation osmolarity
(Cadp) as shown in Fig.
1 can be approximated by a binomial equation as,
![]() | (7) |
|
Our finding that cell volume decreases continuously as the adaptation
osmolarity increases, while the cytosolic osmolarity increases stepwise
(compare Fig. 10A with
Fig. 10B) implies that
stiffness of the cell against its osmotic swelling increases stepwise at each
critical osmolarity as the adaptation osmolarity increases. To quantify the
stiffness, the idea of the bulk modulus was introduced for the
Paramecium cells adapted to different osmolarities. The bulk modulus
of the Paramecium cell adapted to a Cadp,
MB(Cadp), is defined as,
![]() | (8) |
![]() | (9) |
The number of osmolytes in the cytosol of a single cell, N, is
assumed to be the same among cells adapted to a Cadp
varied within an osmolarity range where no critical osmolarity is included,
since no osmolyte transport mechanism(s) is activated by varying
Cadp in this range.
Ccyt(Cadp), therefore, can be written
as,
![]() | (10) |
Ccyt(Cadp) in each of three
different osmolarity ranges was calculated according to
equation 10 and shown as three
gray lines with different gray scales in
Fig. 9A (also in
Fig. 10B), i.e. light gray for
the 075 mosmol l-1 range, medium gray for the 75160
mosmol l-1 range and dark gray for the 160204 mosmol
l-1 range. N value for a 075 mosmol l-1
range and that for a 75160 mosmol l-1 range were estimated
as a product of v(75) x 75 mosmol l-1 and that of
v(160) x 160 mosmol l-1, respectively. N
for a 160204 mosmol l-1 range was estimated as a product of
v(160) x 235 mosmol l-1. It is clear from the figure
that Ccyt gradually increases as Cadp
increases within each osmolarity range, since v decreases as
Cadp increases (compare
Fig. 10B with
Fig. 10A). The values for
cyt(Cadp) can be estimated according to
van't Hoff's formula for a diluted solution as,
![]() | (11) |
Each red solid circle on the thick black line in
Fig. 10A shows the natural
cell volume at each corresponding critical osmolarity,
vN(CN). According to the definition of
the natural cell volume, the cytosolic osmolarity of a cell adapted to a
CN, Ccyt(CN),
equals CN. The thick line in
Fig. 10A corresponding to
v(Cadp)
(equation 7), therefore, can be
regarded as a line that corresponds to the natural cell volume as a function
of the cytosolic osmolarity, vN(Ccyt),
and can be written as,
![]() | (12) |
As is shown in Fig. 10B,
Ccyt changes (in mosmol l-1) from 65 to 75
when Cadp changes from 0 to 75 and from
142 to 160
when Cadp changes from 75-160. Changes in
vN corresponding to these changes in
Ccyt are depicted in
Fig. 10A as two thick red
lines superimposed behind the black line for
v(Cadp).
The values for the bulk modulus of cells adapted to different osmolarities were then calculated according to Equations 8 in consideration of a change in vN due to a change in Ccyt (Equation 12; Fig. 10A, red lines), and plotted against Cadp in Fig. 10D. It is clear from the figure that the bulk modulus increases stepwise at each critical osmolarity as the adaptation osmolarity increases. It is also seen that the bulk modulus gradually increases as the cytosolic osmolarity increases within each osmolarity range. These facts imply that the bulk modulus of Paramecium is primarily dependent on the cytosolic osmolarity.
A major cytosolic osmolyte in Paramecium is KCl
(Akita, 1941;
Stock et al., 2002
). It is,
therefore, highly probable that the bulk modulus of the Paramecium
cell increases as cytosolic KCl concentration increases. Cytosolic KCl
concentration might modify the elastic property of the cytoskeleton and its
associated pellicular membrane. The concentration effect of cytosolic KCl on
the mechanical properties and electron microscopical structures of the
cytoskeleton of Paramecium could be examined to try to understand the
mechanism by which the cell's bulk modulus is modified when changing the
adaptation osmolarity, although such changes might be too subtle to be
recognized by conventional fine structure analysis.
Factors that activate the osmolyte transport mechanisms
Decreasing the external osmolarity
The volume of 84 mosmol l-1-adapted cells increased to a maximum
value of 1.15 times that of its initial value after the cells were
subjected to 24, 44 and 64 mosmol l-1, respectively, then began to
decrease (Fig. 5B). The time
needed to reach the maximum volume was shorter when the degree of decrease in
the external osmolarity was larger. These results would seem to imply that RVD
is activated when the cell is swollen to a certain extent (1.15) independently
of the rate of swelling of the cell.
Conversely, the volume of 144 mosmol l-1-adapted cells increased
to a plateau value of 1.20 times that of its initial value and remained
unchanged after reducing the external osmolarity to 84 mosmol l-1
(Figs 2B,
5A). RVD was not activated even
though the cells were swollen to an extent larger than 1.15. Furthermore, the
164 mosmol l-1-adapted cells showed RVD when they were swollen to
an extent of
1.07. i.e. smaller than 1.15, by decreasing the external
osmolarity to 104 msomol l-1
(Fig. 2A). These results imply
that an osmotic swelling of a cell to a critical size is not the primary
factor for activation of RVD. These results also exclude the possibility that
a decrease in the concentration of a certain chemical(s) in the cytosol by
osmotic swelling is the primary factor for activation of RVD.
A plausible factor for activation of RVD is an increase in the cytosolic pressure to a critical extent. As is previously mentioned, the cytosolic pressure is generated as the cell is osmotically swollen. To examine this possibility, the cytosolic pressure of cells showing their maximum swelling, after their subjection to a decreased osmolarity, was estimated by using the data from eight experiments as shown in Figs 2 and 5. Estimated values for the cytosolic pressure are shown in Table 1 together with values for the parameters needed for the estimation.
|
As is shown in Table 1, the
cytosolic pressure of cells showing maximum volume after their subjection to a
decreased osmolarity (Pcyt2) was larger than
1.5x105 Pa, when they showed RVD (+ in the RVD column).
Conversely, Pcyt2 was smaller than
1.5x105 Pa, when the cells did not show RVD ( in
the RVD column). It can, therefore, be said that an increase in the cytosolic
pressure over a threshold value of
1.5x105 Pa is a
plausible factor for activation of RVD.
This threshold pressure is in a range of so called `micro-pressure'
(Macdonald and Fraser, 1999),
that is far lower than a hydrostatic pressure that causes disintegration of
cytoskeletal filamentous structures
(Macdonald, 2001
). Finding
cellular structures or molecules responsible for sensing this small change in
the cytosolic pressure and activating the osmolyte transport mechanism would
be interesting and important.
Increasing the external osmolarity
Fig. 6B shows that the
volume of 64 mosmol l-1-adapted cells decreased to 0.90, 0.80
and 0.65 times that of their initial volume after their subjection to 84, 104
and 124 mosmol l-1, respectively. Cell volume, then began to
increase, i.e. RVI was activated. The rate and the degree of decrease in cell
volume were larger when the degree of increase in the external osmolarity was
larger.
Conversely, as is shown in Fig.
6A, the volume of 4 mosmol l-1-adapted cells decreased
to 0.80 and 0.75 times that of their initial volume after their
subjection to 44 and 64 mosmol l-1, respectively. Even though the
rate and the degree of decrease in cell volume were comparable with or even
larger than those for 64 msomol l-1-adapted cells after their
subjection to an increased osmolariy (Fig.
6B), RVI was not activated. These results imply that neither the
rate nor the degree of decrease in cell volume is the primary factor for
activation of RVI. These results also exclude the possibility that RVI is
activated when cytosolic concentration of a certain chemical(s) reaches a
threshold value as cell volume decreases.
As was previously mentioned, RVI is activated when the external osmolarity is increased beyond a critical osmolarity. This increase in the external osmolarity causes a decrease in cell volume to an extent that is smaller than the natural cell volume, i.e. an osmotic shrinkage of the cell, since the external osmolarity exceeds the cytosolic osmolarity.
Conversely, when an increase in the external osmolarity takes place within an osmolarity range where no critical osmolarity is included, no osmotic shrinkage occurs, although a decrease in cell volume takes place, since the external osmolarity never exceeds the cytosolic osmolarity. It can, therefore, be said that a slackening of the cytoskeleton-associated membrane surrounding the cell by an osmotic shrinkage of the cell, or a cytosolic pressure that is generated as the cell shrinks osmotically and antagonizes the osmotic pressure difference between the cytosol and the external solution is a plausible factor for activation of RVI. As a matter of fact, an indentation on the cell surface, i.e. osmotic shrinkage of the cell, was always observed when the external osmolarity was increased beyond a critical osmolarity and RVI took place (Fig. 4D, a white arrowhead).
Factors that deactivate the osmolyte transport mechanisms
The fact that the cytosolic osmolarity of Paramecium changes
stepwise as the adaptation osmolarity changes
(Stock et al., 2001) implies
that the hypothetical osmolyte transport mechanism responsible for RVD remains
active until the cytosolic osmolarity decreases to the next lower step level.
Several hours are needed for this osmotic adaptation of the cell
(Ishida et al., 1996
).
Conversely, only 2080 min were needed for restoration of cell volume
(Fig. 2A,C,D) This implies that
the cytosolic pressure decreases below the threshold for activation of the
transport mechanism in this period of time, while the mechanism remains
active. It can, therefore, be said that deactivation (or inactivation) of the
transport mechanism is most likely to be cytosolic osmolarity dependent.
Similarly, the hypothetical osmolyte transport mechanism responsible for
RVI remains active until the cytosolic osmolarity increases to the next higher
level. Several hours are needed for this osmotic adaptation of the cell
(Ishida et al., 1996).
Conversely, only 40100 min were needed for restoration of cell volume
(Fig. 3A,C,D). This implies
that the cell recovers from its shrinkage in this period of time, while the
transport mechanism remains active. It can, therefore, be said that
deactivation (or inactivation) of the transport mechanism is most likely to be
cytosolic osmolarity dependent.
K+ channels are involved in the regulatory volume control mechanisms
Neither RVD nor RVI took place in the presence of 10 mmol l-1
TEA+ (Fig. 7A,C).
Furthermore, an increase in KCl concentration in the external solution to 30
mmol l-1 inhibited RVD, so that cell volume continued to increase
after a cell's subjection to a decreased osmolarity
(Fig. 7B). By contrast, the
presence of 30 mmol l-1 KCl in the external solution enhanced RVI,
so that recovery of cell volume from its temporary shrinkage caused by the
cell's subjection to an increased osmolarity took place faster than that in
the presence of normal 2 mmol l-1 KCl
(Fig. 7D). These results
strongly support the idea that K+ channels in the plasma membrane
are involved in cell volume control mechanisms in Paramecium cells.
Involvement of several kinds of K+ channels in regulatory cell
volume control has been demonstrated in several kinds of cells
(Montrose-Rafizadeh and Guggino,
1990; Tang et al.,
2004
).
How much does the contractile vacuole complex participate in the control of cell volume?
It is well known that the rate of fluid discharge from the CV,
(Naitoh et al., 1997a
),
increases when the external osmolarity decreases, while it decreases when the
external osmolarity increases (Kitching,
1967
). Conversely, the volume of an adapted cell remains unchanged
even though the CV discharges its fluid content to the cell exterior. This
implies that an amount of fluid discharged from the CV (fluid outflow) is
somehow supplied to the cell (fluid inflow). If this fluid
outflowinflow balance has been kept during changes in the external
osmolarity, a change in
will have no
affect on an osmotic change in cell volume. However, if a change in the
external osmolarity affects only the outflow, a change in
will have an
affect on the osmotic change in cell volume. That is, an osmotic swelling of
the cell upon decreasing the external osmolarity is buffered by an increase in
, while an
osmotic shrinkage of the cell upon increasing the external osmolarity is
buffered by a decrease in
, since the
inflow does not change.
We estimated how much an osmotic change in cell volume is buffered by a
change in by
using data shown in Figs 2,
3 and
8. The basic assumption for
this estimation is that the fluid inflow remains unchanged until cell volume
reaches its peak or plateau value after changing the external osmolarity. As
is shown in Table 2, when the
cells were subjected to a 60 mosmol l-1 decrease in the external
osmolarity (from 164 to 104 mosmol l-1 or from 144 to 84 mosmol
l-1),
50% (67% and 43%) of the water that
entered the cell osmotically was discharged through the CV until the cell
volume reached its highest value. Conversely, when the cells were subjected to
a 60 mosmol l-1 increase in the external osmolarity (from 104 to
164 mosmol l-1 or from 84 to 144 mosmol l-1),
20%
(27% and 18%) of the osmotically expelled water was compensated
for by the CVC until the cell volume reached its lowest value. It is,
therefore, conceivable that an immediate change in
after changing
the external osmolarity buffers a large initial osmotic change in cell volume,
so that the cell will not be subjected to mechanical disruption. In their
physiological studies on the contractile vacuole function of Tetrahymena
pyriformis, Dunham and his colleagues had earlier suggested a role for
the CV in buffering the osmotic changes in cell volume
(Stoner and Dunham, 1970
;
Dunham and Kropp, 1973
, see
also Patterson, 1980
).
|
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Acknowledgments |
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Footnotes |
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On leave of absence from Institute of Basic Medical Sciences, University of
Tsukuba, Tenno-Dai 1-1-1, Tsukuba 305-8575, Japan
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References |
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---|
Akita, Y. K. (1941). Electrolytes in Paramecium. Memoir. Fac. Sci. Agric. Taihoku Imp. Univ. 13,99 -120.
Allen, R. D. and Fok, A. K. (1988). Membrane dynamics of the contractile vacuole complex of Paramecium. J. Protozool. 35,63 -71.
Allen, R. D. and Naitoh, Y. (2002). Osmoregulation and contractile vacuoles of protozoa. Int. Rev. Cytol. 215,352 -394.
Baumgarten, C. M. and Feher, J. J. (2001). Osmosis and regulation of cell volume. In Cell Physiology Source Book, A Molecular Approach, 3rd edn (ed. N. Sperelakis), pp.319 -355. San Diego, San Francisco, New York, Boston, Sydney, Tokyo: Academic.
Dunham, P. B. and Kropp, D. L. (1973). Regulation of solute and water in Tetrahymena. In Biology of Tetrahymena (ed. A. M. Elliot), pp.165 -198. Dowden, Stroudsburg, PA, USA: Dowden, Hutchinson & Ross.
Fok, A. K. and Allen, R. D. (1979). Axenic Paramecium caudatum. I. Mass culture and structure. J. Protozool. 26,463 -470.[Medline]
Hoffmann, E. K. and Dunham, P. B. (1995). Membrane mechanisms and intracellular signalling in cell volume regulation. Int. Rev. Cytol. 161,173 -262.[Medline]
Ishida, M., Aihara, M. S., Allen, R. D. and Fok, A. K.
(1993). Osmoregulation in Paramecium: the locus of fluid
segregation in the contractile vacuole complex. J. Cell
Sci. 106,693
-702.
Ishida, M., Fok, A. K., Aihara, M. S. and Allen, R. D.
(1996). Hyperosmotic stress leads to reversible dissociation of
the proton pump-bearing tubules from the contractile vacuole complex in
Paramecium. J. Cell Sci.
109,229
-237.
Jepps, M. W. (1947). Contribution to the study of the sponges. Proc. R. Soc. Lond. B Biol. Sci. 134,408 -417.
Kitching, J. A. (1967). Contractile vacuoles, ionic regulation and excretion. In Research in Protozoology, vol. 1 (ed. T. T. Chen), pp.307 -336. London, UK: Pergamon.
Lang, F., Bush, G. L., Ritter, M., Volkl, H., Waldegger, S.,
Gulbins, E. and Haussinger, D. (1998). Functional
significance of cell volume regulatory mechanisms. Physiol.
Rev. 78,247
-306.
Macdonald, A. G. (2001). Effects of high pressure on cellular processes. In Cell Physiology Source Book, A Molecular Approach 3rd edn (ed. N. Sperelakis), pp.1003 -1023. San Diego, San Francisco, New York, Boston, London, Sydney, Tokyo: Academic.
Macdonald, A. G. and Fraser, P. J. (1999). The transduction of very small hydrostatic pressures. Comp. Biochem. Physiol. A. 122,13 -36.[CrossRef]
Montrose-Rafizadeh, C. and Guggino, W. B. (1990). Cell volume regulation in the nephron. Annu. Rev. Physiol. 52,761 -772.[CrossRef][Medline]
Naitoh, Y., Tominaga, T., Ishida, M., Fok, A. K., Aihara, M. S.
and Allen, R. D. (1997a). How does the contractile vacuole of
Paramecium multimicronucleatum expel fluid? Modelling the expulsion
mechanism. J. Exp. Biol.
200,713
-721.
Naitoh, Y., Tominaga, T. and Allen, R. D.
(1997b). The contractile vacuole fluid discharge rate is
determined by the vacuole size immediately before the start of discharge in
Paramecium multimicronucleatum. J. Exp. Biol.
200,1737
-1744.
Patterson, D. J. (1980). Contractile vacuole and associated structures: their organization and function. Biol. Rev. 55,1 -46.
Stock, C., Allen, R. D. and Naitoh, Y. (2001).
How external osmolarity affects the activity of the contractile vacuole
complex, the cytosolic osmolarity and the water permeability of the plasma
membrane in Paramecium multimicronucleatum. J. Exp.
Biol. 204,291
-304.
Stock, C., Grønlien, H. K., Allen, R. D. and Naitoh,
Y. (2002). Osmoregulation in Paramecium: in
situ ion gradients permit water to cascade through the cytosol to the
contractile vacuole. J. Cell Sci.
115,2339
-2348.
Stoner, L. C. and Dunham, P. B. (1970). Regulation of cellular osmolarity and volume in Tetrahymena. J. Exp. Biol. 53,391 -399.[Medline]
Strange, K. (1994). Cellular and Molecular Physiology of Cell Volume Regulation. Boca Raton, FL, USA: CRC.
Tang, X. D., Santarelli, L. C., Heinemann, S. H. and Hoshi, T. (2004). Metabolic regulation of potassium channels. Annu. Rev. Physiol. 66,131 -159.[CrossRef][Medline]