Osmoregulation in Paramecium: in situ ion gradients permit water to cascade through the cytosol to the contractile vacuole

Osmoregulation in Paramecium multimicronucleatum is based on an intricate interplay between the fluid segregation activity of the contractile vacuole complex (CVC), the regulatory mechanisms that control the cytosolic osmolarity and the water permeability of the plasma membrane(Stock et al., 2001). By accumulating and expelling the excess cytosolic water that enters the cell osmotically from the exterior, the CVC keeps the cytosolic osmolarity constant independently of the osmolarity of the external solution as long as the external solution remains hypotonic to the cytosol. However, in cells that are long-term adapted to external osmolarities equal to or higher than the cytosolic osmolarity, the cytosolic osmolarity will be shifted to a higher level, allowing the cells to continue to acquire water at these higher external osmolarities, and thus for the CVC systems to resume cycling and to maintain their fluid segregation activity.

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.

Cells of Paramecium multimicronucleatum (syngen 2)(Allen and Fok, 1988) were grown in axenic culture medium at 24°C(Fok and Allen, 1979) and harvested at the late logarithmic phase. The cell density was 4-6×10⁶ cells 1^-1. Cell cultures of 12 ml each were centrifuged at 80 g for 25 seconds so that the cells formed a loose pellet. The cells were suspended in an adaptation solution (see below) and centrifuged again into a loose pellet. This washing procedure was repeated twice and the cells were finally suspended in 5 ml of this solution. The cells were kept in this adaptation solution for more than 18 hours prior to experimentation. All experiments were performed at room temperature(25-27°C).

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, R_CVC, in buffer-free solutions of set A. R_CVC 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.

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^-1CaCl₂ instead of 2.0 mmol l^-1 KCl. Other ionic components were the same as those in the solutions of the second set (B).

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 4.5μm.

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^-1by 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^-1K⁺, respectively.

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.

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.

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 ×63 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, R_CVC,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 R_CVC 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.

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.

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.

Table 1 shows K⁺,Na⁺, Ca²⁺ and Cl^- activities in both the in vivo cytosol and the in vivo CV fluid of P. multimicronucleatum under standardized saline conditions, (in mmol l^-1) 2.0 KCl, 0.25 CaCl₂, 20 sorbitol and 1.0 Mops-KOH buffer (pH 7.0). Paramecium cells were adapted to this solution for 18 hours. The most abundant ions were K⁺ and Cl^-, in both the cytosol and the CV fluid, while Na⁺, carried over from earlier culture conditions, was present in only low amounts. This was true even in adaptation solutions containing 2.0 mmol l^-1 NaCl instead of 2.0 mmol l^-1 KCl (Na⁺ activity was 3.3±0.9 mmol l^-1 (n=10) in the cytosol and 4.8±1.2 mmol l^-1 (n=5) in the CV). We, therefore, focused on K⁺ and Cl^- in the present paper and will provide detailed information about Na⁺ activities in a subsequent study. As expected, cytosolic Ca²⁺ levels were below the sensitivity of the Ca²⁺-selective electrode, while, in the CV fluid, Ca²⁺was present in only trace amounts. The K⁺ and Cl^-activities in the CV fluid were 2.5 to 2.4 times higher, respectively, than their activities in the cytosol (Table 2).

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^-1solutions 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.

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=2.7×10^-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).

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=6.3×10^-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).

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=4.8×10^-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=2.9×10^-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.

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 (compareFig. 3Ai,ii withFig. 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).

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 (compareFig. 3Bii withFig. 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).

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.

As shown in Table 3, the substitution of equimolar choline or Ca²⁺ for 2 mmol l^-1K⁺ caused a marked decrease in RCVC accompanied by a decrease in the cytosolic osmolarity.

When choline was substituted for K⁺, R_CVCwas 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⁺, R_CVC 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.

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. R_CVC and cell volume were continuously monitored after the application of furosemide.

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%.

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 withTable 4).

Fig. 5 shows the time courses of changes in R_CVC(Fig. 5A) and in the cell volume (Fig. 5B) after the application of furosemide. R_CVC 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 R_CVC nor an increase in the cell volume (data not shown).

Under standardized conditions, we found that the in vivo CV fluid is hypertonic to the cytosolic fluid. K⁺ and Cl^- activities in the CV fluid are 2.4-fold higher than they are in the cytosol. This finding is different from earlier studies that concluded, based on micropuncture and freezing-point depression studies, that the CV fluids of Chaos carolinensis (Riddick,1968) and Amoeba proteus(Schmidt-Nielsen and Schrauger,1963) were both hypotonic to the cytosol. A hypertonic CV fluid,as we find in Paramecium, would permit water to flow down its concentration gradient from the cytosol into the CV. However, this raises the question of how so much K⁺ and Cl^- can enter the CV against the uphill concentration gradients of these ions. This question is addressed below.

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, seeDunham 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).

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 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.

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.

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 R_CVC (Table 3).

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). R_CVC 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 R_CVC 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 [∼30×10⁸μm² for the CVC membrane(Tominaga et al., 1998;Tominaga et al., 1999) and∼40×10⁸ μm² for the plasma membrane(Stock et al., 2001)]. The water permeability of these two membranes is also the same[∼0.17×10^-5 μm min^-1 Pa^-1 for the CVC membrane (C. S. et al., unpublished) and 0.18×10^-5μm 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+α) mosmol l^-1(52-22-20=10), where α represents the activity of osmolytes other than K⁺ and Cl^- in the CV fluid. As α 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).

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 R_CVC 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.

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 (compareFig. 3B, part ii withFig. 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, seePlattner 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.

Our finding that CV/cytosol activity ratios of K⁺ and Cl^- in the cell remain constant independently of R_CVC 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.

The authors thank Marilynn S. Aihara for valuable technical support. This work was supported by NSF grant MCB 9809929. We also acknowledge the German Academic Exchange Service, DAAD, for earlier financial support of CS(Stipendium im Rahmen des Gemeinsamen Hochschulsonderprogramms III von Bund und Ländern) and the Norwegian Research Society, NFR, for financial support of H.K.G.