1 Pacific Biomedical Research Center, Snyder Hall 306, University of Hawaii at
Manoa, 2538 The Mall, Honolulu, HI 96822, USA
2 Department of Biology, University of Oslo, PO Box 1051, Blindern, N-0316 Oslo,
Norway
* Author for correspondence (e-mail: stock{at}pbrc.hawaii.edu )
Accepted 15 March 2002
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Summary |
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Key words: Contractile vacuole complex, Ion-selective microelectrode, K+/Cl- activity, Osmoregulation, Paramecium multimicronucleatum
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Introduction |
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Not yet understood are: (1) the mechanisms by which cytosolic water is conveyed to the CVC lumen through the CVC membrane, (2) the mechanisms by which the fluid segregation activity of the CVC responds to a change in the amount of water that enters the cytosol osmotically from the external solution and (3) the mechanisms by which the cytosolic osmolarity increases in response to a hypertonic increase in the external osmolarity.
To understand these mechanisms, it is of vital importance to know the ion
species and concentrations in the in vivo cytosol and in the in vivo
contractile vacuole (CV) fluid, as well as their changes in response to
changes in the external osmotic and ionic environments. Therefore,
conventional liquid ion-exchanger ion-selective microelectrodes for
K+, Na+, Ca2+ and Cl-
(Ammann, 1986) were employed to
measure the in vivo activities of these ions in both the cytosol and the CV
fluid in Paramecium cells under standardized conditions, and in cells
adapted to various osmolarities and ionic conditions. This is the first time
that ion concentrations of the CV of any cell or organism have been measured
directly in an in vivo CV.
We found that K+ and Cl- are the major osmolytes in both the in vivo cytosol and the in vivo CV fluid and that the activity of these ions as well as the overall fluid osmolarities remain higher in the CV than in the cytosol. We therefore propose that K+ and Cl- transporters are present in both the CVC membrane and the plasma membrane, and that the control of these transporters is involved in regulating the fluid segregation activity of the CVC as well as regulating cytosolic osmolarity.
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Materials and Methods |
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Adaptation solutions
We chose to use standard saline as our standard as it has been commonly
used by physiologists to study Paramecium
(Kamada, 1931;
Naitoh and Eckert, 1968
).
The first set of adaptation solutions (set A) consisted of five solutions with different osmolarities: 24, 64, 104, 124 and 164 mosmol 1-1. The osmolarity was adjusted by adding different amounts of sorbitol. Besides the different sorbitol concentrations, the solutions contained (in mmol l-1): 2.0 KCl, 0.25 CaCl2 and 1.0 MOPS-KOH buffer (pH 7.0). The osmolarities of these solutions were measured by using a freezing point depression osmometer (Micro-Osmometer Model 3 MO plus, Advanced Instruments, Norwood MA).
The second set (set B) of adaptation solutions consisted of two solutions each with a different osmolarity (24 and 124 mosmol l-1, adjusted by sorbitol). These solutions contained 2.0 mmol l-1 choline chloride instead of the 2.0 mmol l-1 KCl. Other ionic components were the same as those in the solutions of the first set. However, to keep the solution free of any inorganic monovalent cation, no MOPS buffer was added. In order to exclude any effects caused by the absence of the MOPS buffer we examined the CVC activity, RCVC, in buffer-free solutions of set A. RCVC of cells adapted to a MOPS-free 24 mosmol l-1 solution of set A was 65.1±17.4 fl s-1 (n= 14) and did not differ from that in cells adapted to MOPS-containing 24 mosmol l-1 solution (72.8±13.1 fl s-1; n=9; Table 3). Thus, the effect of removing the MOPS buffer from these solutions was negligible.
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The third set (set C) of adaptation solutions consisted of two solutions each with a different osmolarity (24 and 124 mosmol l-1, adjusted by sorbitol). These solutions contained 1.25 mmol l-1 CaCl2 instead of 2.0 mmol l-1 KCl. Other ionic components were the same as those in the solutions of the second set (B).
Ion-selective microelectrodes
Double-barrel borosilicate glass capillaries with filaments (1.5 mm in
outer and 0.84 mm in inner diameter; World Precision Instruments, Sarasota,
FL) were pulled using a horizontal micropipette puller (Model P-97, Sutter
Instrument Company, Novato, CA) to obtain double-barrel microcapillary
pipettes. Their overall outer tip diameters varied between 3 and 4.5
µm.
The inside glass wall of one barrel of a two-barrelled micropipette was
silanized (Deitmer and Munsch,
1995). A small amount (<1 µl) of 5% tributylchlorosilane
(Fluka Chemical, Milwaukee, WI) in carbon tetrachloride (Mallinckrodt Chemical
Works, St Louis, MO) was introduced into the tip of the barrel. The pipette
was then baked on a hot plate for 5 minutes at 460-500°C. After cooling,
the tip of the measuring electrode was filled with a small amount of liquid
K+, Cl-, Na+ or Ca2+ exchange
resin (World Precision Instruments, Sarasota, FL). The backing solutions for
the resin-containing barrels were either 100 mmol l-1 KCl or NaCl
or CaCl2 for either the K+- and Cl- or the
Ca2+-selective electrodes, respectively.
The non-silanized barrel of the microcapillary pipette was filled with
lithium acetate solution and served as the reference electrode. To minimize
the tip potential of this barrel, the ionic strength of the lithium acetate
was adjusted to approximate that of the cytosol. Based on the assumption that
the cytosolic osmolarity is dependent mostly on monovalent electrolytes, we
used 35, 85 and 125 mmol l-1 lithium acetate to measure cells
adapted to external osmolarity ranges of 24-64, 104-124 and 164 mosmol
l-1, respectively, as the cytosolic osmolarities of cells adapted
to those external osmolarities were approximately 70, 170 and 250 mosmol
l-1, respectively (Stock et
al., 2001). A silver chloride-coated silver wire (0.25 mm thick)
was put into each barrel to conduct the electrical potential difference
between the two barrels to an operational amplifier (INA-114, Burr-Brown,
Tucson, AZ) for recording.
The ion sensitivity and its linearity were tested for each electrode before and after each measurement using one of three different sets of KCl [Fig. 1A (part c), B (part c)], NaCl or CaCl2 calibration solutions of known ionic activity and a calibration curve was drawn showing the relationship between the electrical potential and the ionic activity. The ionic activity range of the calibration solutions was chosen to include the actual ionic activity to be measured. In order to imitate the cytosolic ionic activities, the ionic strengths of these solutions were also adjusted as needed to 70, 170 or 250 mmol l-1 by adding lithium acetate. Partial or complete substitution of NaCl for the lithium acetate in the calibration solution did not make a difference in the calibration curve. Readings of the K+ activities obtained with Na+ present in the calibration solutions hardly differed from those obtained from solutions without Na+: -0.34±0.53 mmol l-1 (n=5) and -1.6±2.19 mmol l-1 (n=5) in solutions containing 5 and 30 mmol l-1 K+, respectively.
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The data obtained for cytosol and CV fluids were analyzed only when the ion sensitivity of the electrode as well as its linearity were the same before and after the measurement.
Determination of ion concentrations in the cytosol and the CV
A minute amount of adaptation solution containing an adapted cell was
introduced into a droplet of mineral oil on a coverslip. The cell was
immobilized by removing excess adaptation solution through a suction pipette.
The tip of an ion-selective microelectrode was then inserted into either the
cytosol or into the CV (Fig.
1C) where it was kept for several seconds to obtain a stable
potential difference corresponding to the ionic activity of the compartment
[Fig. 1A (part b), B (part b)]. The ionic activity was obtained from the calibration curve
[Fig. 1A (part c), B (part c)].
When the double-barreled electrode was inserted into the CV, the CV continued
to accumulate fluid normally but fluid discharge was blocked.
Determination of the fluid segregation activity by the CVC,
RCVC
The experimental procedure for this experiment was identical to that
previously described (Stock et al.,
2001). An experimental chamber was filled with a 0.02%
poly-L-lysine solution. Cells suspended in an adaptation solution were
introduced into the chamber at one end, while the poly-L-lysine solution was
removed from the chamber at the other end by absorption with filter paper. If
necessary, a solution exchange was performed by this same means. Cells that
adhered to the chamber were used for experimentation.
Images of the CVs of adhered cells obtained using Nomarski microscope optics (Leitz x63 objective, Leica Mikroskop. u. Sys. GmbH, Wetzlar, Germany) were video-recorded (ERG-6300, Panasonic Industrial, Secaucus, NJ) through a CCD camera (CCD-72, DAGE MTI, Michigan City, IN) together with the signals of a video timer (FOR. A. Japan). On replayed images of the CV, the period of time between two successive fluid discharges and the maximum diameter of the CV immediately before the start of fluid discharge were measured. The rate of fluid expulsion by the CVC, RCVC, was calculated by dividing the maximum volume of the CV immediately before fluid discharge (calculated from the diameter of the CV based on the assumption that the rounded CV is spherical) by the time that had elapsed since the last fluid discharge. Only one CV in each cell was measured and its RCVC evaluated.
To measure the effects of furosemide (Sigma, St Louis, MO) on the fluid segregation activity, the 24 mosmol l-1 adaptation solution containing 2 mmol l-1 K+ was carefully replaced by 1 mmol l-1 furosemide (final concentration) dissolved in 0.1% DMSO (v/v) in the same 24 mosmol l-1 adaption solution.
Determination of the cytosolic osmolarity
The cytosolic osmolarities of cells adapted for 18 hours to 24 or 124
mosmol l-1 solutions of sets A, B and C were determined according
to the method previously described (Stock
et al., 2001). The method was essentially the same as that
employed by Stoner and Dunham (Stoner and
Dunham, 1970
) except that we used Congo Red and a
spectrophotometer instead of radioactive 14C-inulin and a
scintillation counter for our determinations.
Determination of changes in cell volume
Cells in 24 mosmol l-1 adaptation solution (set A) were
compressed under a coverslip to a thickness of 26 µm using latex beads
with an average diameter of 25.7 µm as spacers between the two coverslips.
The cells were left in this condition for approximately 15 minutes before the
adaptation solution was replaced by a 1 mmol l-1 (final
concentration) furosemide-containing 24 mosmol l-1 adaptation
solution or by a 0.1% (v/v) DMSO-containing adaptation solution, which served
as the control. Images of compressed cells, viewed from below, were video
recorded. The area of the coverslip that is covered by the cell is
proportional to the cell volume. It was measured on replayed video images
using NIH Image 1.62. The change in cell volume was expressed in percent
change in pixel numbers that were covered by the cell. This was converted into
cell area. The edge of the cell not touching a coverslip was ignored, as it
was assumed to be essentially the same before, during and after the volume
change. The significance of all data was tested using the Mann-Whitney
U-test (P<0.05). Values are presented as
means±s.e.m.
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Results |
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K+ and Cl- activities in cells adapted to
increasing osmolarities
K+ and Cl- activities were determined in both the
cytosol and the CV fluid of P. multimicronucleatum cells that had
been adapted for 18 hours to 24, 64, 104, 124 or 164 mosmol l-1
solutions all containing the same ionic compositions. In addition,
K+ and Cl- activities were also determined in cells
adapted to 24 or 124 mosmol l-1 solutions where an equimolar
choline chloride or CaCl2 concentration was substituted for the 2
mmol l-1 KCl. Hereafter, these solutions will be called choline- or
Ca2+-containing solutions.
Cells adapted to K+-containing solutions
Cytosol
As shown in Fig. 2A (open
circles), the cytosolic K+ activities of cells adapted to external
osmolarities of 24 or 64 mosmol l-1 were 22.6±7.7 mmol
l-1 (mean±s.d., n=7) or 21.2±5.8 mmol
l-1 (n=11), respectively. They did not differ
significantly (P=0.69; t-test). The K+ activities
in cells adapted to 104 mosmol l-1 (62.1±13.7 mmol
l-1; n=8) or 124 mosmol l-1 (60.3±18.9
mmol l-1; n=8) were nearly the same (P=0.5).
However, the K+ activity in cells adapted to 104 mosmol
l-1 was significantly higher than that in cells adapted to 64
mosmol l-1 (P=2.7x10-5), and the
K+ activity in cells adapted to 164 mosmol l-1
(83.9±16.7 mmol l-1; n=9) was significantly higher
than that in cells adapted to 124 mosmol l-1
(P=0.017).
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Fig. 2B (open circles) shows that the cytosolic Cl- activities of cells adapted to 24 or 64 mosmol l-1 were 27.3±5.9 mmol l-1 (n=9) or 28.0±2.5 mmol l-1 (n=8), respectively. They did not differ significantly (P=0.76). The Cl- activities in cells adapted to 104 mosmol l-1 (69.2±4.8 mmol l-1; n=7) or 124 mosmol l-1 (66.7±5.5 mmol l-1; n=7) did not differ either (P=0.38). However, the Cl- activity in cells adapted to 104 mosmol l-1 was significantly higher than that in cells adapted to 64 mosmol l-1 (P=6.3x10-9), and the Cl- activity in cells adapted to 164 mosmol l-1 (100.2±21.5 mmol l-1; n=5) was significantly higher than that in cells adapted to 124 mosmol l-1 (P=0.02).
CV fluid
As shown in Fig. 2A (closed
circles), the K+ activities in the CV fluid of cells adapted to 24
mosmol l-1 (56.0±2.8 mmol l-1; n=5) or
64 mosmol l-1 (50.3±8.2 mmol l-1; n=5)
did not differ significantly (P=0.2). The K+ activities in
cells adapted to 104 (132.3±2.9 mmol l-1; n=5) or
124 mosmol l-1 (140.6±17.8 mmol l-1;
n=5) were also nearly the same (P=0.36). The K+
activity in the CV fluid of cells adapted to 104 mosmol l-1,
however, was significantly higher than that of cells adapted to 64 mosmol
l-1 (P=4.8x10-6), and the K+
activity in the CV fluid of cells adapted to 164 mosmol l-1
(176±28.8 mmol l-1; n=5) was significantly higher
than that of cells adapted to 124 mosmol l-1
(P=0.027).
Fig. 2B (closed circles) shows that the Cl- activities in the CV fluid of cells adapted to 24 mosmol l-1 (66.5±8.3 mmol l-1; n=6) or 64 mosmol l-1 (69.7±8.6 mmol l-1; n=5) did not differ significantly (P=0.55). The Cl- activities in cells adapted to 104 mosmol l-1 (134.6±14.8 mmol l-1; n=7) or 124 mosmol l-1 (131.4±10.4 mmol l-1; n=7) did not differ either (P=0.66). However, the Cl- activity in the CV fluid of cells adapted to 104 mosmol l-1 was significantly higher than that of cells adapted to 64 mosmol l-1 (P=2.9x10-6), and the Cl- activity in the CV fluid of cells adapted to 164 mosmol l-1 (193.6±21.8 mmol l-1; n=5) was significantly higher than that of cells adapted to 124 mosmol l-1 (P=0.0016).
The K+ and Cl- activities in the CV fluid were always approximately 2.3 (2.1-2.5) and 2.2 (1.9-2.5) times, respectively, more than those in the cytosol (Table 2). The activity ratios were calculated by dividing the value for an ionic activity in the CV fluid by the corresponding value for the ionic activity of the same ion in the cytosol.
Cells adapted to choline-containing solutions
The K+ and Cl- activities in both the cytosol and the
CV fluid were much lower in the choline-containing medium than in the
K+-containing medium (compare
Fig. 3Ai,ii with
Fig. 3Ci,ii). The K+
activities in the cytosol were 5.6±1.2 mmol l-1
(n=6) or 20.1±1.5 mmol l-1 (n=6), while
those in the CV fluid were 13.6±1.8 mmol l-1 (n=6)
or 49.7±6.0 mmol l-1 (n=6) in cells adapted to 24
or 124 mosmol l-1, respectively. The Cl- activities in
the cytosol were 11.5±1.1 mmol l-1 (n=6) or
33.1±4.4 mmol l-1 (n=7), while those in the CV
fluid were 24.0±2.2 mmol l-1 (n=7) or
66.0±9.5 mmol l-1 (n=5) in cells adapted to 24 or
124 mosmol l-1, respectively. The activity ratios between the CV
fluid and the cytosol were 2.4 and 2.5 for K+ and 2.1 and 2.0 for
Cl- in cells adapted to 24 and 124 mosmol l-1,
respectively (Table 2).
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Cells adapted to Ca2+-containing solutions
The K+ activities in both the cytosol and the CV fluid of cells
adapted to Ca2+-containing solutions
(Fig. 3Bi) were much lower than
those of cells adapted to K+-containing solutions
(Fig. 3Ci), whereas the
Cl- activities of cells adapted to Ca2+- containing
solutions did not differ that much from those of cells adapted to
K+-containing solutions (compare
Fig. 3Bii with
Fig. 3Cii). The K+
activities in the cytosol were 8.4±1.6 mmol l-1
(n=9) or 8.3±1.5 mmol l-1 (n=9), while
those in the CV fluid were 18.9±3.9 mmol l-1 (n=8)
or 20.0±4.3 mmol l-1 (n=5) in cells adapted to 24
or 124 mosmol l-1, respectively. The Cl- activities in
the cytosol were 17.1±4.6 mmol l-1 (n=6) or
60±4.2 mmol l-1 (n=5), while those in the CV fluid
were 86.0±14.1 mmol l-1 (n=6) or 117.3±8.6
mmol l-1 (n=5) in cells adapted to 24 or 124 mosmol
l-1, respectively. The activity ratios between the CV fluid and the
cytosol were 2.3 and 2.4 for K+ and 5.0 and 2.0 for Cl-
in cells adapted to 24 and 124 mosmol l-1, respectively
(Table 2).
Ca2+ activities in the CV fluid
Because of their innate limit of sensitivity, Ca2+-selective
microelectrodes could not be used to detect Ca2+ in the cytosol.
However, the Ca2+ activity in the CV fluid could be detected. As
shown in Fig. 4, in
K+-containing solutions the Ca2+ activities in the CV
fluid were 0.23±0.13 mmol l-1 (n=5) and
0.7±0.3 mmol l-1 (n=5) in cells adapted to 24 and
124 mosmol l-1. These concentrations were slightly higher in cells
adapted to solutions containing choline instead of K+, i.e.
0.7±0.4 mmol l-1 (n=5) in 24 mosmol
l-1-adapted cells and 1.2±0.5 mmol l-1
(n=5) in 124 mosmol l-1-adapted cells. However, the
Ca2+ activities were remarkably higher in the CV fluid of cells
adapted to Ca2+-containing solutions. The activities were
15.4±5 mmol l-1 (n=5) and 29.7±8.3 mmol
l-1 (n=6) in cells adapted to 24 and 124 mosmol
l-1, respectively.
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Effects of K+-deficiency in the external solution on the
rate of fluid segregation by a CVC (RCVC) and the cytosolic
osmolarity
As shown in Table 3, the
substitution of equimolar choline or Ca2+ for 2 mmol l-1
K+ caused a marked decrease in RCVC accompanied by a
decrease in the cytosolic osmolarity.
When choline was substituted for K+, RCVC was reduced by more than 50% in cells adapted to 24 mosmol l-1 and by more than 70% in cells adapted to 124 mosmol l-1, while the cytosolic osmolarities were reduced by 50% and by approximately 30%, respectively. When Ca2+ was used as substitute for K+, RCVC was decreased by more than 40% in cells adapted to 24 mosmol l-1 and by more than 60% in cells adapted to 124 mosmol l-1, while the cytosolic osmolarities were decreased by more than 55% and by approximately 21%, respectively.
Effects of the external application of furosemide
Cells adapted to a 2 mmol l-1 K+-containing 24 mosmol
l-1-solution were exposed to the same adaptation solution
containing 1 mmol l-1 furosemide (final concentration) initially
dissolved in DMSO (final concentration 0.1% v/v). Furosemide has been used as
an inhibitor of K+ and Cl- transport (for references see
Discussion). K+ and Cl- activities were determined 10-20
minutes after cell exposure to furosemide. RCVC and cell
volume were continuously monitored after the application of furosemide.
K+ activities and Cl- activities
As shown in Table 4, the
K+ activity in the cytosol decreased by 54% from its control value
measured after only the solvent was applied (0.1% v/v DMSO in the
K+-containing 24 mosmol l-1 adaptation solution), while
the K+ activity in the CV fluid decreased by 57% from its control
value. The Cl- activity in the cytosol decreased by 52%, while that
in the CV fluid decreased by 55%.
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The ratios for the K+ and Cl- activities between the CV fluid and the cytosol did not change in the presence of furosemide. The ratio for K+, as well as that for Cl-, was approximately 2.5 in cells that were not exposed to the drug and 2.4 in cells that were exposed to the drug (Table 2). The exposure of cells to 0.1% DMSO (v/v) only did not have any effect on the K+ and Cl- activities in either the cytosol or the CV fluid (compare Table 1 with Table 4).
RCVC and cell volume
Fig. 5 shows the time
courses of changes in RCVC
(Fig. 5A) and in the cell
volume (Fig. 5B) after the
application of furosemide. RCVC decreased within 5 minutes
of the application of furosemide from its control value of 89.7±35.4 to
37.2±31.2 fl s-1 (n=5), while the cell volume
increased in 15 minutes by 10.1±4.4% (n=5) over its original
volume. The addition of solvent only, 0.1% DMSO (v/v) dissolved in the
K+-containing 24 mosmol l-1 adaptation solution, caused
neither a decrease in RCVC nor an increase in the cell
volume (data not shown).
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Discussion |
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KCI is a major osmolyte in the cytosol
The presence of an ample amount of K+ together with
Cl- in the cytosol indicates that KCl is potentially the major
osmolyte that causes the cytosolic osmolarity to be hypertonic to the external
solution. The sum of the cytosolic K+ and Cl- activities
accounted for 67, 66 and 70% of the total cytosolic osmolarity in cells
adapted to 24-64, 104-124 and 164 mosmol l-1, respectively. These
percentages were obtained by multiplying the K+ activity by 2 (to
account for equimolar amounts of Cl-) and dividing this product by
the corresponding cytosolic osmolarity (66, 185 and 240 mosmol l-1,
respectively) (Stock et al.,
2001).
For P. caudatum adapted to external osmolarities of less than 64
mosmol l-1 Akita (Akita,
1941) obtained cytosolic K+ concentrations ranging from
17.2 to 28.3 mmol l-1 using a titration method, Yamaguchi
(Yamaguchi, 1963
) obtained
values ranging from 16.2 to 22.0 mmol l-1 by using flame
spectrophotometry, and, more recently, Oka et al.
(Oka et al., 1986
) obtained a
value of 21 mmol l-1 by using an atomic absorption method. Our
value of 22 mmol l-1 for P. multimicronucleatum cells
adapted to 24-64 mosmol l-1 solutions containing K+
obtained by using K+-selective microelectrodes is consistent with
these values. In Tetrahymena pyriformis, the cytosolic K+
concentration ranges from 22 to 44 mmol l-1, depending on the
culture medium used (for a review, see
Dunham and Kropp, 1973
).
In P. multimicronucleatum, the remaining 30-34% of the cytosolic
osmolarity not accounted for by KCl may consist in part of other inorganic
ions present in smaller amounts, such as Na+
(Table 1), and in part by a
variety of charged organic compounds. Free amino acids, such as glycine,
alanine and proline, have been found to act as osmolytes in Miamiensis
avidus (Kaneshiro et al.,
1969), Tetrahymena pyriformis
(Stoner and Dunham, 1970
) and
P. calkinsi (Cronkite et al.,
1993
; Cronkite and Pierce,
1989
).
K+ and Cl- activities in the cytosol and in the
in vivo CV fluid increase in a stepwise fashion as the external osmolarity
increases linearly
As shown in Fig. 2, both
K+ and Cl- activities in the cytosol of P.
multimicronucleatum increase in a stepwise fashion as the external
osmolarity is increased linearly by the addition of sorbitol. These stepwise
increases reflect the similar stepwise increases reported to occur in the
overall cytosolic osmolarity when the external osmolarity approaches or
exceeds the cytosolic osmolarity (Stock et
al., 2001).
K+ and Cl- are present in the in situ CV fluid and their activities, which are always higher in the CV than in the cytosol, increase in the same stepwise fashion as the cytosolic K+ and Cl- activities (Fig. 2). This result indicates that K+ and Cl- are transported from the cytosol into the CV where they are concentrated.
The CV fluid is always hypertonic to the cytosol
The sum total of the K+ plus the Cl- activities in
the CV fluid is approximately 120, 264 or 365 mmol l-1 in cells
adapted to external osmolarities of 24-64, 104-124 or 164 mosmol
l-1, respectively. The cytosolic osmolarities in cells adapted to
these three osmolarity ranges are 66, 185 and 240 mosmol l-1,
respectively (Stock et al.,
2001). Thus, the osmolarity of the CV fluid is always hypertonic
to the cytosol. This fact supports the idea that cytosolic water is
osmotically conveyed to the CVC lumen.
A hypothesis for cellular osmoregulation in Paramecium
Based on our findings, we propose a hypothesis that K+ and
Cl- ions are co-transported from the external solution into the
cytosol to keep the cytosol hypertonic to the external solution. The resulting
osmotic gradient across the plasma membrane will allow water to enter the cell
osmotically. At the same time, the CV fluid is kept hypertonic to the cytosol
by the activity of K+ and Cl- transport systems in the
CVC membrane, rather than by bicarbonate-transport, as was proposed earlier
(Tominaga et al., 1998).
Excess cytosolic water, therefore, can flow osmotically into the CVC lumen.
The building up of gradients will allow water to cascade from the exterior of
the cell into the cytosol across the plasma membrane and then across the CVC
membrane into the CVC. We conclude that this is the basis for keeping the cell
volume constant under natural environmental conditions.
Support for the hypothesis that K+ and Cl- are
the major osmolytes for osmoregulation in Paramecium
Transporting K+ and Cl- into the cytosol from
the external solution is needed to maintain fluid segregation activity by the
CVC
The presence of K+ and Cl- in the CV fluid
(Fig. 2,
Fig. 3) implies that the
cytosolic K+ and Cl- will be constantly expelled to the
exterior of the cell. To maintain a normal fluid segregation activity,
K+ and Cl- should be constantly resupplied to the
cytosol. It is unlikely that K+ and Cl- can be
reabsorbed from the CV fluid into the cytosol through the CV membrane before
the fluid is expelled as (1) many measurements of the ion activities in the CV
fluid were obtained just before fluid discharge and (2) slight increases in
K+ ionic activities were detectable when the tips of ionselective
microelectrodes were placed outside the cell adjacent to the pore as the CV
was discharging (data not shown). Thus, under standard ionic conditions the
cytosolic K+ and Cl- are assumed to be constantly
supplied from the external solution through the plasma membrane. This idea is
supported by our findings that cells adapted to solutions deprived of
K+ show a significant decrease in the overall cytosolic osmolarity
as the cytosolic K+ and Cl- activities
[Table 3,
Fig. 3A (parts i,ii), B (parts
i,ii)] decrease. At a given external osmolarity a decrease in the
overall cytosolic osmolarity will lead to a decrease in the osmotic gradient
across the plasma membrane and, therefore, to a reduction of
RCVC (Table
3).
Effects of furosemide on K+ and Cl- activities
and on cell volume
Furosemide is known to inhibit the reabsorption of Na+,
K+ and Cl- in the ascending limb of the loop of Henle
(Brater, 1998), the
[Na+, K+, Cl-] co-transport and the
[K+, Cl-] co-transport in mammalian erythrocytes
(Lauf, 1984
;
Canessa et al., 1986
;
Garay et al., 1988
). In
Paramecium, the primary effect of furosemide should, therefore, be
essentially the same as that of eliminating K+ from the external
solution. Adding furosemide to the external medium resulted in a reduction of
both the K+ activity and the Cl- activity in the cytosol
(Table 4).
RCVC decreased as the K+ and Cl-
activities in the cytosol and in the CV fluid decreased
(Fig. 5A). Furosemide
presumably inhibits the K+ and Cl- transport through the
plasma membrane. Whether furosemide can directly reduce the K+ and
Cl- transport across the CVC membrane is not known, but is unlikely
as the K+ and Cl- activity ratios between the cytosol
and the CV fluid are unaltered in cells treated with furosemide
(Table 2). The cell volume
temporarily increased, while RCVC decreased after the cell
was subjected to furosemide (Fig.
5). This implies that the osmotic water flow into the cell across
the plasma membrane exceeds the water flow into the CVC. In a
Paramecium cell, the total membrane area of the CVC is almost the
same as that of the plasma membrane [
30x108
µm2 for the CVC membrane
(Tominaga et al., 1998
;
Tominaga et al., 1999
) and
40x108 µm2 for the plasma membrane
(Stock et al., 2001
)]. The
water permeability of these two membranes is also the same
[
0.17x10-5 µm min-1 Pa-1 for
the CVC membrane (C. S. et al., unpublished) and 0.18x10-5
µm min-1 Pa-1 for the plasma membrane
(Stock et al., 2001
)].
Furosemide, therefore, may cause the osmotic gradient across the CVC membrane
to be lower than that across the plasma membrane. Upon exposure to furosemide,
the sum of K+ and Cl- activities decreased from 46 to 22
mmol l-1 in the cytosol and from 117 to 52 mmol l-1 in
the CV fluid (Table 4). If we
assume that the cytosolic osmolytes other than K+ and
Cl- (a total of 66-46=20 mosmol l-1 before furosemide
treatment) cannot change as quickly as the cytosolic K+ and
Cl- activities, the osmotic gradient across the plasma membrane
will be approximately 18 mosmol l-1 (22+20-24=18), while that
across the CVC membrane will be (10+
) mosmol l-1
(52-22-20=10), where
represents the activity of osmolytes other than
K+ and Cl- in the CV fluid. As
can be assumed to
be a small fraction of the total CV osmolarity, the total osmotic gradient
across the CVC membrane should be smaller than that across the plasma
membrane. The cell will consequently swell, as was observed
(Fig. 5B).
The CV/cytosol ratios for K+ activity and Cl-
activity remain constant independently of RCVC
In cells adapted to osmolarity ranges of 24 to 164 mosmol l-1,
the ratio for K+-activity varied in a narrow range of 2.1-2.5 and
that for Cl- activity varied between 1.9-2.5
(Table 2), whereas
RCVC decreased from approximately 100 to 10 fl
s-1 (Stock et al.,
2001). The ratio for the K+ activity (
2.3) and
that for the Cl- activity (
2.2) are nearly equal. This may
indicate that much of the K+ and Cl- are, indeed,
co-transported by a single transporter in the CVC membrane.
KCl is not a major osmolyte in the cytosol under
K+-deficient conditions
The percentage of KCl in the overall cytosolic osmolytes reached only 35%
and 31% in cells adapted to choline chloride-containing 24 and 124 mosmol
l-1 solutions, respectively
(Fig. 3A,
Table 3), compared with 67% and
66% in cells adapted to K+ containing 24 and 124 mosmol
l-1 solutions, respectively
(Fig. 3C,
Table 3). As in other cells,
choline may be actively transported from the surrounding solution through the
plasma membrane into the cytosol, although a choline transporter has not yet
been identified in the plasma membrane of Paramecium. Choline
transporters have been found in a variety of cell species from cells of the
central nervous system (Knipper et al.,
1991; Laganiere et al.,
1991
) to yeast (Li et al.,
1991
) and even in membranes of organelles such as the inner
membranes of rat liver mitochondria
(Porter et al., 1992
). In
bacteria such as Escherichia coli and Bacillus subtilis,
choline is taken up and converted to betaine, which is then used as
osmoprotectant in high osmolarity environments
(Kempf and Bremer, 1998a
;
Kempf and Bremer, 1998b
).
When CaCl2 was substituted for the external KCl, the percentage
of KCl in the overall cytosolic osmolytes was 56% and 11% in cells adapted to
24 and 124 mosmol l-1, respectively
(Fig. 3B,
Table 3). The cytosolic
Cl- activity was two times and seven times larger than the
cytosolic K+ activity in cells adapted to 24 and 124 mosmol
l-1, respectively. This implies that there must be counter-cations
for Cl- other than K+ present in the cytosol.
Ca2+ can not be a counter-cation for Cl- in the cytosol,
because of its low cytosolic activity, less than 10-7 mol
l-1 (Naitoh and Kaneko,
1972; Nakaoka et al.,
1984
; Machemer,
1989
), a figure that is related to its diverse functions as second
messenger and regulatory ion. Arginine and lysine as well as oligopeptides
with an alkaline isoelectric point may be plausible candidates as the
counter-cations for Cl- in a pH-neutral cytosol.
In contrast to a marked decrease in K+ activity in the CV fluid, the Cl- activity in the CV fluid decreased little or even increased following Ca2+ substitution for the external K+ (Fig. 3B, part ii). It increased by 23% in cells adapted to 24 mosmol l-1 and decreased by 11% in cells adapted to 124 mosmol l-1 (compare Fig. 3B, part ii with Fig. 3C, part ii). This indicates that only a small fraction of the Cl- ions are acting as counter-anions for K+ ions in the CV fluid of these cells, and that, as in the cytosol, there must be cations other than K+ in the CV fluid.
As shown in Fig. 4, the
Ca2+ activity in the CV fluid markedly increased under
Ca2+-containing, K+-deficient conditions. This implies
that Ca2+ is actively transported from the cytosol into the CVC
lumen to keep the CV fluid hypertonic to the cytosol. In fact, the presence of
CaCl2 in the CV fluid can account for 35% and 59% of the
Cl- activity in the CV fluid of cells adapted to 24 and 124 mosmol
l-1, respectively. Thus, under conditions of Ca2+
stress, the CVC can play a role in the extrusion of excess cytosolic
Ca2+ that would be deleterious to many Ca2+-dependent
intracellular signaling systems. Ca2+ can enter the cytosol from
the external solution via stimulus-activated Ca2+ channels in the
plasma membrane and can also be released from proven intracellular
Ca2+ storage sites such as alveoli (for a review, see
Plattner and Klauke, 2001).
Moniakis et al. (Moniakis et al.,
1999
) report a Ca2+-ATPase in Dictyostelium
discoideum that is localized to the membranes of the CV in this cell. The
gene expression for this Ca2+-ATPase is upregulated when cells are
grown in Ca2+-rich medium. In addition, the authors postulate that
a proton gradient generated by the V-type H+-ATPase facilitates the
Ca2+ transport into the CV.
Further considerations
Our finding that CV/cytosol activity ratios of K+ and
Cl- in the cell remain constant independently of
RCVC imply that water molecules are stoichiometrically
transported into the CVC lumen in association with the K+ and/or
the Cl- transport. A stoichiometry can be obtained by dividing 55.6
(molar concentration of water) by the K+ activity in the CV fluid.
The numbers of water molecules per a single K+ transport are
approximately 990, 400 and 300 in cells adapted to osmolarity ranges of 24-64,
104-124 and 164 mosmol l-1, respectively. The stoichiometry
decreases as the cytosolic osmolarity or the cytosolic K+ activity
increases.
The stoichiometry of water transport by the CVC membrane differs from that
of the human Na+/glucose co-transporter (SGLT1) that functions as a
molecular water pump, where the stoichiometry is constant and independent of
both the Na+ and the glucose concentration
(Loo et al., 1996;
Meinild et al., 1998
).
A fundamental property of animal cells is the ability to regulate their own
cell volume. Under hypotonic stress, cells readjust their volume after
transient osmotic swelling by a mechanism known as regulatory volume decrease
(RVD) (for a review, see Okada et al.,
2001). Osmotic swelling causes the activation of solute transport
systems resulting in a loss of osmolytes and a concomitant loss of cell water.
In most cell types, the major osmotically active solute lost during the RVD
response is KCl (Hoffmann and Mills,
1999
).
We have now demonstrated that in Paramecium, a K+ and
Cl- transport-dependent water transport across the CVC membrane is
certainly involved in the cell volume regulation under hypotonic conditions.
The osmoregulatory mechanism in Paramecium might be analogous to RVD
in other cell types. Biagini et al.
(Biagini et al., 2000) report a
hypotonically induced, swelling-activated loss of K+ through the
plasma membrane in the free-living protozoan Hexamita inflata. H.
inflata lacks a CV (Brugerolle,
1974
). Its K+-extrusion system apparently corresponds
to RVD and appears to be a mechanistic alternative to a working CVC
system.
As soon as Paramecium faces hypertonic conditions its osmoregulatory system can be used to restore cell volume. Exposed to a hypertonic condition, the water flow through the plasma membrane will stop or even be reversed. This would result in a decrease in cell volume. The CV activity will then stop and K+ and Cl- will no longer be expelled from the cell. Extracellular K+ and Cl-, however, will continue to be imported into the cytosol. This will eventually lead to a cytosolic osmolarity equaling or exceeding the external osmolarity. At this point, water can again be acquired osmotically, which will cause the cell to swell back to its original volume. Presumably, the CVC will again become active as its ion activity increases above that of the cytosol. Overall, this process reminds one of the regulatory volume increase (RVI) in animal cells.
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